Methods for protein refolding

ABSTRACT

The present invention discloses improved methods of disaggregating protein aggregates, and refolding denatured proteins, using high pressure. In particular, the present invention provides for the use of agitation, high temperature, “stepped” depressurization, dialysis and dilution under pressure to increase the speed and extent of aggregate dissolution and protein refolding.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT/US01/45728, filed Oct. 31,2001, which claims priority from U.S. Provisional Application Ser. No.60/244,808, filed Oct. 31, 2000.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the field of proteinbiochemistry. More particularly, it concerns improved methods for therenaturation and refolding of polypeptides aggregates.

2. Description of Related Art

The aggregation of proteins is of significant concern in thebiotechnology, pharmaceutical and medical communities. In vitro,aggregation is observed in virtually every step in the production,refolding, purification, storage and shipping of protein therapeutics(Carpenter et al., 1997; Clark et al., 1999). In vivo, numerouspathogenic conditions in humans (e.g., Alzheimer's disease, Parkinson'sdisease and prion diseases (Kelly, 1996; Kelly, 1998; Prusiner, 1998;Scherzinger et al., 1999)) have protein aggregation and formation ofinsoluble deposits associated with the condition and, as a result,research on the characterization of the aggregates and mechanisms ofaggregation in these diseases is an active area of medical research.

Aggregation in human protein deposition diseases, which displaysorganization in the form of insoluble fibrils, has brought increasedsignificance to the study of protein misassembly and aggregationprocesses in general (Wetzel, 1999). Investigation into the reversal ofaggregation and precipitation processes has immediate practicalimplications for the production, purification and delivery oftherapeutic proteins.

In the production of therapeutic proteins, aggregated precipitates(e.g., inclusion bodies) is commonly reversed by dissolution of theprecipitated aggregates in the presence of high concentrations ofchaotrope (e.g., 6 M guanidine hydrochloride). Such harsh conditionsresult both in disaggregation (solubilization) and in nearly completeunfolding of the protein. Commonly, refolding is effected by removal ofthe chaotrope via dialysis or dilution to protein concentrations of ca.10 to 50 μg/mL (Clark et al., 1999). Because refolding is commonly afirst-order (in protein concentration) process and aggregation asecond-order or higher process, refolding yields are improved at lowerprotein concentrations (Clark et al., 1999). Soluble aggregates areoften separated from the native protein by costly and time consumingcolumn chromatographies. Separated soluble aggregates are typicallydiscarded, thus reducing overall protein yields and substantiallyincreasing protein production costs. An alternative to chaotropicdissolution to dissolve insoluble aggregates or column purification toremove soluble aggregates is disaggregation by pressure (Foguel et al.,1999; Gorovits & Horowitz 1998; St. John et al., 1999).

Several research groups have exploited the ability of pressure todissociate native protein oligomers (Silva & Weber 1993). In addition,others have explored the use of pressure to disaggregate and refoldproteins from soluble non-native protein aggregates (Foguel et al.,1999; Gorovits & Horowitz, 1998) and precipitated, insoluble non-nativeaggregates (St. John, et al., 1999). Gorovits and Horowitz 1998 usedhigh pressure to inhibit formation of soluble aggregates in 3.9 M ureasolutions of rhodanase, and to reverse the formation of solubleaggregates. However, Gorovits & Horwitz (1998) report that “pressure . .. is not able to reverse large aggregates.” Treatment at 2.4 kbar for 90minutes of soluble aggregates formed from P22 tailspike protein reducedaggregate levels from 41.1 to 17.6% (Foguel et al., 1999).

St. John et al. (1999) used high pressure to dissolve and recover nativeprotein from large, insoluble aggregates at pressures on the order of200 MPa, including aggregates formed as inclusion bodies. High yields athigh protein concentrations of refolded, active human growth hormone,lysozyme and β-lactamase from insoluble, precipitated aggregates wereachieved using non-denaturing concentrations of guanidine hydrochloridein combination with pressure or pressure in the absence of guanidinehydrochloride (St. John et al., 1999). In the specific case of insolubleaggregates of lysozyme containing non-native intermolecular covalentdisulfide bonds that served to crosslink the insoluble precipitates,redox shuffling agents such as mixtures of reduced and oxidizedglutathione were used in combination with high pressure and 0.8Mguanidine HCl to afford high yields of folded, biologically activeprotein. In the specific case of aggregated and precipitated andaggregated human growth hormone, low levels of a chaotrope such asguanidine HCl were used to optimize recovery of soluble, native protein.In the specific case of B-lactamase inclusion bodies, addition ofguanidine HCl did not increase yield of biologically active B-lactamase,but did result in higher solubilization of contaminating proteins.

Nonetheless, improved methods for the high-pressure dissociation ofprotein aggregates and refolding of solubilized proteins are desired.

SUMMARY OF THE INVENTION

Thus, in accordance with the present invention, improved methods for theuse of high pressure in protein disaggregation and refolding areprovided. In one embodiment, there is provided a method of producingbiologically active disaggregated protein from protein aggregatescomprising:

-   -   (i) providing a protein aggregate;    -   (ii) mixing said protein aggregate with a reducing agent in        amount sufficient to reduce disulfide bonds therein;    -   (iii) subjecting the mixture of step (ii) to increased pressure,        as compared to ambient pressure, whereby said protein aggregate        dissolves;    -   (iv) dialyzing the mixture under pressure, whereby said reducing        agent is removed and disulfide bonds reform; and    -   (v) removing the dissolved protein from increased pressure,        whereby said protein refolds such that biological activity is        retained. The method may further comprise the step of agitating        said protein aggregate and/or dissolved protein to enhance        dissolution and/or refolding; or may further comprise the step        of subjecting said protein aggregate, prior to refolding, to a        temperature of about 30° C. to about 125° C. The method may        optionally exclude a chaotropic agent. Suitable reducing agents        include diothiothreitol, glutathione, dithioerythritol, or        β-mercaptoethanol. The increased pressure may comprise about 500        atmospheres to about 10,000 atmospheres.

The steps (ii)-(v) may be performed in about 3 hours to about 12 hours,particular in about 6 hours. Steps (ii) and (iii) may be performed inthe presence of a chaotropic agent, and said method may further comprisethe step of removing said chaotropic agent. Suitable chaotropic agentsinclude guanidine, guanidine sulfate, guanidine hydrochloride, urea,thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate.

The protein aggregate may comprise inclusion bodies, soluble andinsoluble precipitates, soluble non-native oligomers, gel, fibrils,films, filaments, protofibrils, amyloid deposits, plaques, or dispersednon-native intracellular oligomers. The protein aggregate also may besubjected to pressure at high concentration, for example, at aconcentration is from about 5 to 20 mg/ml, or more particularly, about10 mg/ml. The protein aggregate may then be diluted under pressure, forexample, to about 1 mg/ml.

In another embodiment, there is provided a method of producingbiologically active disaggregated protein from protein aggregatescomprising:

-   -   (i) providing a protein aggregate;    -   (ii) subjecting the said protein aggregate to increased        pressure, as compared to ambient pressure, and agitation,        whereby said protein aggregate dissolves; and    -   (iii) removing the dissolved protein from increased pressure,        whereby said protein refolds such that biological activity is        retained. The method may further comprise the step of agitating        the dissolved protein to enhance refolding. Agitation may be        provided by ultrasound energy, mechanical stirring, shaking, or        pumping through static devices. It may may further comprise        subjecting said protein aggregate, prior to refolding, to a        temperature of about 30° C. to about 80° C. Optionally, the        method does not utilize a chaotropic agent. The increased        pressure may comprise about 500 atmospheres to about 10,000        atmospheres.

The steps (ii)-(v) may be performed in about 3 hours to about 12 hours,particular in about 6 hours. Steps (ii) and (iii) may be performed inthe presence of a chaotropic agent, and said method may further comprisethe step of removing said chaotropic agent. Suitable chaotropic agentsinclude guanidine, guanidine sulfate, guanidine hydrochloride, urea,thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate.

The method may further comprising mixing said protein aggregate with areducing agent in amount sufficient to reduce disulfide bonds therein;and further may comprise dialyzing said mixture under pressure, wherebysaid reducing agent is removed and disulfide bonds reform. The reducingagent may be removed by diafiltration or ultrafiltration, or it may benegated by addition of an oxidizing agent. Suitable reducing agent isdiothiothreitol, glutathione, dithioerythritol, or β-mercaptoethanol.

The protein aggregate may comprise inclusion bodies, soluble andinsoluble precipitates, soluble non-native oligomers, gel, fibrils,films, filaments, protofibrils, amyloid deposits, plaques, or dispersednon-native intracellular oligomers. The protein aggregate also may besubjected to pressure at high concentration, for example, at aconcentration is from about 5 to 20 mg/ml, or more particularly, about10 mg/ml. The protein aggregate may then be diluted under pressure, forexample, to about 1 mg/ml.

In yet another embodiment, there is provided a method of producingbiologically active disaggregated protein from protein aggregatescomprising:

-   -   (i) providing a protein aggregate;    -   (ii) subjecting the said protein aggregate to increased        pressure, as compared to ambient pressure, and high temperature        of about 30° C. to about 125° C., whereby said protein aggregate        dissolves; and    -   (iii) removing the dissolved protein from increased pressure and        high temperature,        whereby said protein refolds such that biological activity is        retained. Optionally, the method does not include use of a        chaotropic agent. The method may further comprise the step of        agitating said protein aggregate and/or dissolved protein to        enhance dissolution and/or refolding; or may further comprise        mixing said protein aggregate with a reducing agent in amount        sufficient to reduce disulfide bonds therein. The mixture may be        dialyzed under pressure, whereby said reducing agent is removed        and disulfide bonds reform. Alternatively, the reducing agent I        removed by diafiltration or ultrafiltration, or negated by        addition of an oxidizing agent. Suitable reducing agents include        diothiothreitol, glutathione, dithioerythritol, or        βmercaptoethanol. The increased pressure may comprise about 500        atmospheres to about 10,000 atmospheres.

The steps (ii)-(v) may be performed in about 3 hours to about 12 hours,in particular in about 6 hours. Steps (ii) and (iii) may be performed inthe presence of a chaotropic agent, and said method may further comprisethe step of removing said chaotropic agent. Suitable chaotropic agentsinclude guanidine, guanidine sulfate, guanidine hydrochloride, urea,thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate.

The protein aggregate may comprise inclusion bodies, soluble andinsoluble precipitates, soluble non-native oligomers, gel, fibrils,films, filaments, protofibrils, amyloid deposits, plaques, or dispersednon-native intracellular oligomers. The protein aggregate also may besubjected to pressure at high concentration, for example, at aconcentration is from about 5 to 20 mg/ml, or more particularly, about10 mg/ml. The protein aggregate may then be diluted under pressure, forexample, to about 1 mg/ml.

In still yet another embodiment, there is provided a method of producingbiologically active disaggregated protein from protein aggregatescomprising:

-   -   (i) providing a protein aggregate;    -   (ii) mixing said protein aggregate with a reducing agent in        amount sufficient to reduce disulfide bonds therein;    -   (iii) subjecting the mixture of step (ii) to increased pressure,        as compared to ambient pressure, high temperature of about        30° C. to about 125° C., and agitation, whereby said protein        aggregate dissolves;    -   (iv) removing or neutralizing said reducing agent, whereby        disulfide bonds reform; and    -   (v) removing the dissolved protein from increased pressure and        high temperature,        whereby said protein refolds such that biological activity is        retained. Optionally, the method does not include use of a        chaotropic agent. The method may further comprise dialyzing said        mixture under pressure, whereby said reducing agent is removed        and disulfide bonds reform; or the method may further comprise        removing said reducing agent by diafiltration or        ultrafiltration. Alternatively, the effect of said reducing        agent is negated by addition of an oxidizing agent. Suitable        reducing agents include diothiothreitol, glutathione,        dithioerythritol, or β-mercaptoethanol. The increased pressure        may comprise about 500 atmospheres to about 10,000 atmospheres.

Steps (ii)-(iii) may be performed in about 1 hour to about 12 hours, inparticular, in about 6 hours. The protein aggregate may compriseinclusion bodies, soluble and insoluble precipitates, soluble non-nativeoligomers, gel, fibrils, films, filaments, protofibrils, amyloiddeposits, plaques, or dispersed non-native intracellular oligomers. Theprotein aggregate also may be subjected to pressure at highconcentration, for example, at a concentration is from about 5 to 20mg/ml, or more particularly, about 10 mg/ml. The protein aggregate maythen be diluted under pressure, for example, to about 1 mg/ml.

Steps (ii) and (iii) may be performed in the presence of a chaotropicagent, and said method further comprises the step of removing saidchaotropic agent. Suitable chaotropic agents include guanidine,guanidine sulfate, guanidine hydrochloride, urea, thiocyanate, sarcosyl,sodium dodecyl sulfate, or sodium octyl sulfate.

In a further embodiment, there is provided a method of producingbiologically active disaggregated protein from protein aggregatescomprising:

-   -   (i) providing a protein aggregate;    -   (ii) subjecting the said protein aggregate to a first increased        pressure, as compared to ambient pressure, whereby said protein        aggregate dissolves;    -   (iii) subjecting the dissolved protein to a second pressure that        is less than said first increased pressure, but still increased        pressure as compared to ambient pressure, and    -   (iv) removing the dissolved protein from said second increased        pressure,        whereby said protein refolds such that biological activity is        retained. The first increased pressure may be about 200 MPa to        about 1000 MPa. The second increased pressure may be about 100        MPa. The method may further comprise agitation of the protein        under pressure; or the method may further comprising subjecting        said protein to high temperature, for example a high temperature        of about 30° C. to about 125° C.

The method may further comprise mixing the protein aggregate, prior toincreased pressure, with a reducing agent. The method may also comprisedialyzing said mixture under pressure, whereby said reducing agent isremoved and disulfide bonds reform. Alternatively, the reducing agent isremoved by diafiltration or ultrafiltration, or is negated by additionof an oxidizing agent. Reducing agents include diothiothreitol,glutathione, dithioerythritol, or β-mercaptoethanol.

The protein aggregate also may be subjected to pressure at highconcentration, for example, at a concentration is from about 5 to 20mg/ml, or more particularly, about 10 mg/ml. The protein aggregate maythen be diluted under pressure, for example, to about 1 mg/ml.

Steps (ii)-(iv) may be performed in about 3 hours to about 12 hours, ormore particularly, in about 6 hours. Optionally, the method excludeschaotropic agents. Steps (ii)-(iv) may be performed in the presence of achaotropic agent, and said method would further comprise the step ofremoving said chaotropic agent. Suitable chaotropic agents includeguanidine, guanidine sulfate, guanidine hydrochloride, urea,thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate.

The protein aggregate may comprise a protein multimer, for example, ahetero-multimer or a homo-multimer. Multimers may be selected from thegroup consisting of a dimer, a trimer, a tetramer, a pentamer, ahexamer, a heptamer, an octamer and a nonamer. Examples of multimericproteins include interferon-γ, hemoglobin, lactic acid dehydrogenase,antibodies and antibody fragments.

All of the preceding methods may be performed on soluble denaturedprotein as well.

In additional embodiments, there are provided:

-   -   a method of rendering a protein aggregate susceptible to        dissolution by chaotropes, detergents and/or increased        temperature comprising subjecting said protein aggregate to high        pressure in combination with one or more of chaotropes,        detergents and/or increased temperature;    -   a method increasing the shelf-life of a protein sample        comprising the steps of removing soluble protein aggregates by        applying high pressure, followed by depressurization;    -   a method of screening a protein composition for refolding        conditions comprising:        -   (i) providing a protein composition in physically distinct            replicate samples;        -   (ii) subjecting said replicate samples to different            conditions comprising high pressure and varying temperature,            buffers of varying pH, buffers of varying salt            concentration, varying protein concentration, varying            reducing agent concentration, varying stabilizing agents,            varying chaotropic agents, varying detergents, varying            surfactants; and        -   (iii) removing said replicate samples from high pressure;            and        -   (iv) assessing protein refolding;    -   a method of inactivating virus in a sample containing an protein        of interest comprising:        -   (i) providing a sample containing a desired protein, said            protein being in a native or non-native state;        -   (ii) treating said sample to reduce or eliminate infectious            virus particles therein; and        -   (iii) subjecting said sample to a high pressure protein            refolding procedure;    -   and a method of producing biologically active disaggregated        protein from protein aggregates comprising:        -   (i) providing a protein aggregate;        -   (ii) subjecting the mixture of step (i) to increased            pressure, as compared to ambient pressure, high temperature            of about 30° C. to about 125° C., and agitation, whereby            said protein aggregate dissolves;        -   (iii) altering the pH of the mixture of step (ii) by            dialysis; and        -   (iv) removing the dissolved protein from increased pressure            and high temperature,    -   whereby said protein refolds such that biological activity is        retained.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: High pressure recovery of rhGH aggregates in both buffer aloneand 0.75M GdmHCl. Atmospheric standards are labeled with open symbols,while pressurized samples are labeled with solid symbols. Refoldingbuffers contain no GdmHCl.

FIGS. 2A-2B: TEM micrographs of rhIFN-γ (FIG. 2A) thermally-inducedaggregates and (FIG. 2B) guanidine hydrochloride-induced aggregates.Frames A1 and B are at a magnification of 40,000 (Bar=200 μm) and A2 at80,000 (Bar=100 μm).

FIG. 3: Mass percent concentration versus EM diameter as measured byGEMMA for native (thick line) and thermally-induced aggregates (thinline) of rhIFN-γ. (A) EM diameter region for monomer (ca. 4.4 nm) anddimer (ca. 5.5 nm) and (B) EM diameter region for aggregates. Note thatthe particle size distribution is split into two windows for clarity andthat the two windows have different mass percent scales. The responsefor native rhIFN-γ in the 7 to 10 nm range (window B) is within thenoise level of the instrument.

FIG. 4: Second derivative UV spectra of rhIFN-γ thermally-induced (□)and guanidine hydrochloride-induced (▴) aggregates prior to pressuretreatment. Included with the spectra of aggregates are rhIFN-γ liquidcontrol at 0.1 MPa (solid line) and pressure-dissociated at 250 (X) MPa.The extremum near 286 nm in the native and pressure-dissociated spectrawas used to follow the refolding event after dissociation of aggregates.Except for the 250 MPa spectrum, all spectra were collected at 0.1 MPa.

FIG. 5: Area normalized second derivative FTIR spectra ofthermally-induced aggregates (□) and guanidine hydrochloride-inducedaggregates (▴) prior to pressure treatment. The spectrum of nativerhIFN-γ is included for comparative purposes and is represented as asolid line. All spectra were collected at 0.1 MPa.

FIGS. 6A-6B: FIG. 6A—Absorbance at 310 run versus time for guanidinehydrochloride-induced aggregates at 250 MPa in succinate buffer. Thetotal rhIFN-γ concentration was 1.0 mg/mL and the guanidinehydrochloride concentration in solution was ca. 5 mM. The absorbance ofnative rhIFN-γ at 310 nm in the absence of aggregates is ca. 0.06 AUmL/cm mg (data not shown). FIG. 6B—The second derivative extremum (near286 nm) height versus time at 100 MPa for rhIFN-γ (1 mg/mL) guanidinehydrochloride-induced (▴) and thermally-induced (□) aggregatesdissociated at 250 MPa. Error bars of are 95% confidence intervals onthe spectral height. Spectral heights for the pressure-dissociated format 250 MPa (dashed line), native liquid control (solid line with X) andpressure-refolded after equilibration at 0.1 MPa (dotted line) areincluded on the plot for reference.

FIGS. 7A-7C: TEM micrographs of pressure-treated (FIG. 7A)thermally-induced aggregates, (FIG. 7B) guanidine hydrochloride-inducedaggregates and (FIG. 7C) native control rhIFN-γ (no pressure treatment)at magnifications of 40,000. Short fibrous structures were observed inall samples, but the dominant structure was amorphous (e.g., A2).Bar=200 μm.

FIGS. 8A-8B: Second derivative UV spectra of (FIG. 8A) thermally-inducedand (FIG. 8B) guanidine hydrochloride-induced pressure-treatedaggregates. All spectra were collected at 0.1 MPa immediately aftercompletion of the pressure refolding protocol. Refolding was performedat 20 mg/mL (solid symbols), 10 mg/mL (open symbols) and 1 mg/mL (dottedline) and spectra collected immediately after dilution to ca. 1 mg/mL.The spectrum of rhIFN-γ native control (solid line) is superimposed inboth windows for reference.

FIG. 9: Area normalized second derivative FTIR spectra ofpressure-treated (FIG. 9A) thermally-induced and (FIG. 9B) guanidinehydrochloride-induced aggregates. All spectra were collected at 0.1 MPawithin three hours of the completion of the pressure refolding protocol.FTIR spectra were recorded for refolding performed at 20 mg/mL (solidsymbols), 10 mg/mL (open symbols). The spectrum of rhIFN-γ nativecontrol (solid line) is superimposed in both windows for reference.

FIG. 10: Second derivative UV spectra of thermally-induced,pressure-treated aggregates after 2 weeks at 4° C., 0.1 MPa and 1 mg/mL.Refolding was performed at 20 mg/mL (solid symbols), 10 mg/mL (opensymbols) and 1 mg/mL (dotted line) and samples immediately diluted toca. 1 mg/mL. The spectrum of rhIFN-γ native control (solid line) issuperimposed for reference.

FIG. 11: Effect of redox conditions at various pressures on refoldingyield. Conditions were 16 hours of refolding at 25° C., with 6 hours ofdepressurization. Maximum yield was obtained for samples in 4 mM GGSG, 2mM DTT and held at 2000 bar.

FIG. 12: Effect of temperature on refolding yield. Conditions were 2000bar for 24 hours of refolding, 4 mM GGSG, 2 mM DTT, pH 7.2, with 8 hoursof depressurization. Maximum yield was obtained when the refoldingtemperature was held at 25° C.

FIG. 13: Effect of pressure on refolding yield. Conditions were 16 hoursof refolding, 4 mM GGSG, 2 mM DTT, 22° C., pH 7.2, with 8 hours ofdepressurization. Refolding yield was found to be a maximum at 2000 bar.

FIG. 14: Effect of temperature on a VEGF refolding. Conditions were 2000bar for 16 hours, slow depressurization. A yield of 29% was achieved forsamples held at 50° C. Error bars denote 95% confidence level.

FIG. 15: Solubilized Protein from GCSF inclusion bodies: Circles (●)represent atmospheric samples. Squares (▪) represent samples incubatedat 2000 bar for 24 hours.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

Although not visible, soluble aggregates propose an extremely difficultand hazardous problem when producing protein products. Solubleaggregates decrease product values and, in the case of pharmaceuticalpreparations, can lead to dangerous anaphylactic reactions in patientsthat can be fatal. Many processing steps can cause formation of solubleaggregates, including: filtration, ultrafiltration, extraction,precipitation, crystallization, spray/freeze drying, concentration, andchromatography. Because filtration can cause additional formation ofsoluble aggregates, removal of these aggregates is extremely complex anddifficult. In fact, commercially available protein products often aregraded on the content of soluble aggregates in the formulation, and thevalue of a product can be drastically improved by a relatively smallreduction in soluble aggregates (2-5%). Human serum albumin is anexcellent example of a product whose value can be increased by reductionof aggregates.

Likewise, insoluble precipitates of non-native protein are often formedduring the production, purification, packaging, storage, shipping anddelivery of proteins. These aggregates are a human health hazard in thecase of parentally-administered products, because the can provoke severeimmune responses, including anaphylactic shock. Furthermore, synthesisof human proteins in recombinant cell cultures often results in theformation of insoluble precipitates of non-native, aggregated proteintermed inclusion bodies. It should be noted that the inclusion bodiesare often large enough to be viewed by transmission electron microscopy,and may contain covalent non-native intermolecular disulfide bonds.These inclusion bodies are typically processed by first dissolving themin high concentrations of chaotropic agents such as urea or guanidine,adding reducing agents to break disulfide bonds. After dissolution, thedissolved, unfolded protein is then typically diluted to concentrationsbelow 1 mg/ml, and the urea or guanidine is slowly removed bydiafiltration. Redox conditions are often specified at various timesduring the refolding process to allow native disulfide bonds to reform.The overall process requires large amounts of expensive reagents,equipment for handling large volumes of solutions and expensive wastedisposal steps. In addition, the yields of properly folded, nativeprotein are often less than 50%, and the desired protein is found in adilute solution after refolding, requiring additional, expensiveconcentration steps after refolding is complete.

Previously, the inventors have described their work on the use of highpressure to facilitate disaggregation of insoluble protein aggregates,and to facilitate protein refolding. St. John et al. (1999), and in U.S.Ser. No. 09/350,327, by Randolph et al Combining hydrostatic pressurewith low, non-denaturing concentrations of guanidine hydrochloride,human growth hormone aggregates were disaggregated and properly refoldedprotein was recovered at 100%. At 24° C. and in the absence ofguanidine, recovery of native human growth hormone after pressuretreatment at 2 kbar for 24 hours was only 20%.

In particular, U.S. Ser. No. 09/350,327 by Randolph et al. (incorporatedby reference) provides effective methods for disaggregating andrefolding of denatured, aggregated proteins in solution so that properlyfolded, biologically active protein in solution is recovered in highyield. Dissociation of protein aggregates takes place as the pressure isincreased from about 0.25 kbar up to no more than about 12 kbar. Therefolding takes place at pressures between about 0.25 kbar to about 3.5kbar, advantageously at about 1.5 kbar to about 3 kbar. Typically achaotropic agent is present at a concentration that is not effective fordenaturing protein at atmospheric pressure, and sometimes is absentaltogether. Optionally, oxidation-reduction reagents can be incorporatedin the refolding solution so that native intramolecular disulfide bondscan be formed where that is desired. The method is applicable tosubstantially all proteins, especially for insoluble protein aggregates,inclusion bodies, or abnormal oligomeric (soluble) aggregates.

The present invention extends this work in the following ways:

-   -   agitation of protein at various stages, in particular while the        protein is under pressure, speeds and improves the        disaggregation and refolding process;    -   increasing the temperature during various incubations, including        while the protein is undergoing disaggregation and refolding,        also speeds the process and improves the yield, particularly in        the absence of a chaotrope;    -   removing or neutralizing the reducing agent while under        pressure;    -   dilution of protein to working concentration immediately after        pressurization, which permits smaller volumes for high-pressure        refolding equipment, which provides a substantial cost savings;        and    -   using intermediate pressures during refolding to permit        intramolecular and intermolecular interactions to be “sorted”        independently        Using these elements in combination with the previously        disclosed methods, either individually or in combination with        one another, the present inventors provide improved methods of        protein dissagregation and refolding under pressure, including        significant time savings. The following paragraphs describe        various embodiments of the present invention.

Augmenting conventional refolding processes; transition steps. In manyrefolding processes, there are critical steps at which certain reagentsor conditions are removed or negated. These steps provide opportunitiesfor refolded proteins to either unfold or aggregate and precipitate. Inorder to prevent this from occurring, one may utilize the presentinvention to “bridge” one treatment condition to the next. For example,removal of a detergent or chaotropic agent, or reduction of temperature,which are critical for further processing of a protein sample, creates arisk of aggregation. By subjecting samples to increased pressure whiletransitioning to a detergent or chaotrope free solution, or to roomtemperature, one reduces the chance of precipitation.

Similarly, some reagents or conditions, which are insufficient in and ofthemselves to facilitate disaggregation and/or refolding of proteins,can be augmented by the use of high pressure. While certain uses prosperby the exclusion of chaotropes or detergents, other uses may not beaffected by these relatively harsh treatments. By using these reagentsor conditions in conjunction with high pressure, it is possible toimprove their performance and, in some cases, create an operablerefolding process. Such combinations include pressure+increasedtemperature, pressure+detergent, pressure+chaotrope, etc.

Screening of folding conditions for gene products. Protein productioncan be facilitated by genetically modifying an appropriate host cell soas to cause it to produce a known or unknown proteinaceous product,using techniques well known to those skilled in the art. See Sambrook etal (1989). Unfortunately, these products often are found within the hostcell as insoluble precipitates, and there may be considerable difficultyin recovering properly folded proteins from these precipitates. However,since each protein host cell combination is unique, it often takeslaborious empirical experimentation to determine the appropriaterefolding conditions.

In one aspect of the invention, homogenized samples of cell “paste”containing insoluble precipitates of a desired proteinaceous product, orpartially purified preparations (e.g., by centrifugation to remove celldebris, followed by washing of the precipitate with buffered solutionscontaining surfactants to remove lipids) can be screened for optimaldissolution and folding conditions by placing them in individual samplecontainers. To these containers are also added aqueous solutions whichmay contain various buffers (examples include, but are not limited to,phosphate buffer, borate buffer, carbonate buffer, citrate buffer,HEPES, MEPS), salts (examples include, but are not limited to, thechloride, sulfate, and carbonate salts of sodium, zinc, calcium,ammonium and potassium), solubilizing agents (examples include, but arenot limited to, urea, guanidine hydrochloride, guanidine sulfate andsarcosine), and stabilizing agents (examples include, but are notlimited to, nonionic surfactants such as Tween 20, Tween 40, Tween 80,Brij, preservatives such as benzyl alcohol, and carbohydrates such assucrose, raffinose, hydroxyethyl starch, dextran and trehalose).

A preferred container is a multiple-well (e.g., 96-well) sample holder,where multiple protein samples can be placed in an array of samplewells, with each well possibly containing solutions of different pH,ionic strength, salt type, buffer type, and stabilizing agents.Multiple-well sample holders may be conveniently sealed usingcommercially available self-adhesive plastic covers. The containers, orthe entire multiple-well sample holder, may then be placed in a pressurevessel, such as those commercially available from the Flow InternationalCorp. or High Pressure Equipment Co. The remainder of the interiorvolume of the high-pressure vessel may than be filled with water orother pressure transmitting fluid. The pressure may then be elevated asdescribed in the claims, and dissolution and folding of the proteinsample effected.

Disaggregation and refolding of proteins after viral inactivationprocesses. Viral inactivation processes are often used for human plasma,plasma-derived protein products and for cell culture media. Theseprocesses involve treatment of the solution with heat, high pressurecycling or solvent-detergent mixtures. For any of these methods, aproblem arises in that the same process that inactivates viruses alsocauses protein denaturation and aggregation. Thus, a viral inactivationprotocol often represents a compromise between optimal viralinactivation and optimal retention of desired levels of active protein.The result is that either the inactivation is incomplete, valuableprotein components are lost, or both. The problem further is complicatedby the fact that some of the most valuable proteins in the treatedproduct (e.g., Factor VIII) are some of the most sensitive tostress-induced aggregation.

With the inventors' process, the solution containing viruses can betreated with any of the standard methods employed to inactivate viruses.After this treatment, the solution is then subjected to the inventors'high-pressure disaggregation and refolding processes to increase thelevel of native active protein(s). Furthermore, with the capacity torenature protein damaged during viral inactivation in place, moreeffective. (e.g., longer exposure to high temperatures and/or exposureto higher temperatures) viral inactivation processes could be employed.

Viruses that may be of concern include HIV-1, HIV-2, hepatitis A virus,hepatitis B virus, hepatitis C virus, parvovirus, herpes simplex virus Iand II, Epstein Barr virus, HHV6 and cytomegalovirus. The sample may beplasma, blood, plasma-derived protein products, a protein productderived from cultured human cells, serum, or serum-containing cellculture medium. The high pressure protein refolding procedure furthercomprises one or more treatments including high temperature, chaotropicagent, solubilizing agent, reducing agent, agitation and “stepped”depressurization.

Reduction in levels of soluble aggregation that serves as nuclei orprenuclei for protein precipitation. There are several steps during thepurification, storage and processing of protein products that fosterformation of non-native soluble protein aggregates and/or insolubleprecipitates on non-native protein aggregates. These include filtration,dialysis, chromatography, freeze-thawing, exposure to air bubbles duringprocesses such as filling holding tanks or even final product vials,etc. Removing or reducing these aggregates would reduce the risk of lossof much greater amounts of protein during subsequent processing step dueto precipitation. This is because soluble aggregates often serve asnuclei or prenuclei for precipitation. As such, these species can existat relatively low levels (e.g., 1% of the total protein mass) and stilltrigger rapid precipitation of a large fraction (e.g., >30-50% orhigher) of the total protein population. Precipitation can be triggeredwhen a given processing step foster formation of a level of solubleaggregates that exceeds a threshold and/or by exposure of existingsoluble aggregates to a stress (e.g., filtration) that promotes assemblyto a critical nucleus size. Also, the mere fact of long-term storage canresult in precipitation due to soluble aggregates.

Thus, in one embodiment, these nuclei or prenuclei can be removed simplyby processing a whole cell slurry, or partially purified slurry, usingthe high pressure disaggregation and/or refolding processes of thepresent invention. Alternatively, precipitated protein can be separatedfrom the soluble fraction by filtration, chromatography orcentrifugation, and then refolded using the same high-pressure methods.The elimination (or even reduction in the level) of these aggregatesminimizes the risk for protein precipitation in subsequent processingsteps, and can improve shelf-life of refolding protein preparations.

The various aspects of the invention will be described in detail in thefollowing discussion.

II. Definitions

As used herein, a “protein aggregate” is defined as being composed of amultiplicity of protein molecules wherein non-native noncovalentinteractions and/or non-native covalent bonds such as intermoleculardisulfide bonds hold the protein molecules together. Typically, but notalways, an aggregate contains sufficient molecules so that it isinsoluble. There are also abnormal oligomeric proteins which occur inaggregates in solution. In addition, there is typically (but not always)a display of at least one epitope or region on the aggregate surfacewhich is not displayed on the surface of native, non-aggregated protein.“Inclusion bodies” are a type of aggregate of particular interest, towhich the present invention is applicable.

“Atmospheric” or “ambient” pressure is defined as approximately 15pounds per square inch (psi) or 1 bar.

A “binding partner,” or “ligand,” may be included in a refoldingmixture. A “binding partner” is a compound which specifically binds (orotherwise interacts) with a target protein of interest. “Ligands”include, without limitation, antibodies, receptors, peptides,peptidomimetics, vitamins, cofactors, prosthetic groups, substrates,products, competitive inhibitors, metals and other small or largemolecules. The presence of such a binding partner is especiallyadvantageous in a refolding mixture where that binding partner favors anative conformation of the target protein when it interacts with therefolding target protein.

“Biological activity” of a protein as used herein, means at least 10% ofmaximal known specific activity as measured in an assay that isgenerally accepted in the art to be correlated with the known orintended utility of the protein. For proteins intended for therapeuticuse, the assay of choice is one accepted by a regulatory agency to whichdata on safety and efficacy of the protein must be submitted. A proteinhaving greater than 10% of maximal known specific activity is“biologically active” for the purposes of the invention.

“Denatured,” as applied to a protein in the present context, means thatnative secondary and tertiary structure is disrupted to an extent thatthe protein does not have biological activity.

“Degassing” is defined as the removal of gases dissolved in solutionsused in the present invention. Gas is much more soluble in liquids athigh pressure as compared to atmospheric pressure and, consequently, anygas headspace in a sample will be driven into solution uponpressurization. The consequences are two-fold: the additional oxygen insolution may chemically degrade the protein product, and gas exitingsolution upon depressurization may cause additional aggregation. Thus,samples should be prepared with degassed solutions and all headspaceshould be filled with liquid prior to pressurization.

“Depressurization” is a process of decreasing the pressure, from a highpressure, to a lower pressure (usually atmospheric or ambientpresssure). Depressurization takes place over a predetermined period oftime, ranging from 10 seconds to 10 hours, and may be interrupted at oneor more points to permit optimal refolding at intermediate (but stillincreased compared to ambient) pressure levels. The depressurization orinterruptions may be 1 second, 2 seconds, 5 seconds, 10 seconds, 20seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30 minutes, 60minutes, 2 hours, 3 hours, 4 hours, and 5 hours.

“Heterologous” proteins are proteins which are normally not produced bya particular host cell. Recombinant DNA technology has permitted theexpression of relatively large amounts of heterologous proteins (forexample, growth hormone) from transformed host cells such as E. coli.These proteins are often sequestered in insoluble inclusion bodies inthe cytoplasm and/or periplasm of the host cell. The inclusion bodies orcytoplasmic aggregates contain, at least in part, the heterologousprotein to be recovered. These aggregates often appear as bright spotsunder a phase contrast microscope.

“High pressure,” for the purposes of dissociating protein aggregates,means about 0.25 kbar to about 12 kbar. Specifically contemplatedpressures within this range are 0.30 kbar, 0.35 kbar, 0.40 kbar, 0.50kbar, 0.55 kbar, 0.60 kbar, 0.65 kbar, 0.70 kbar, 0.75 kbar, 0.80 kbar,0.85 kbar, 0.90 kbar, 0.95 kbar, 1.0 kbar, 1.1 kbar, 1.2 kbar, 1.3 kbar,1.4 kbar, 1.5 kbar, 1.6 kbar, 1.7 kbar, 1.8 kbar, 1.9 kbar, 2.0 kbar,2.1 kbar, 2.2 kbar, 2.3 kbar, 2.4 kbar, 2.5 kbar, 2.6 kbar, 2.7 kbar,2.8 kbar, 2.9 kbar, 3.0 kbar, 3.1 kbar, 3.2 kbar, 3.3 kbar, 3.4 kbar,3.5 kbar, 3.6 kbar, 3.7 kbar, 3.8 kbar, 3.9 kbar, 4.0 kbar, 4.1 kbar,4.2 kbar, 4.3 kbar, 4.4 kbar, 4.5 kbar, 4.6 kbar, 4.7 kbar, 4.8 kbar,4.9 kbar, 5.0 kbar, 5.1 kbar, 5.2 kbar, 5.3 kbar, 5.4 kbar, 5.5 kbar,5.6 kbar, 5.7 kbar, 5.8 kbar, 5.9 kbar, 6.0 kbar, 6.1 kbar, 6.2 kbar,6.3 kbar, 6.4 kbar, 6.5 kbar, 6.6 kbar, 6.7 kbar, 6.8 kbar, 6.9 kbar,7.0 kbar, 7.1 kbar, 7.2 kbar, 7.3 kbar, 7.4 kbar, 7.5 kbar, 7.6 kbar,7.7 kbar, 7.8 kbar, 7.9 kbar, 8.0 kbar, 8.1 kbar, 8.2 kbar, 8.3 kbar,8.4 kbar, 8.5 kbar, 8.6 kbar, 8.7 kbar, 8.8 kbar, 8.9 kbar, 9.0 kbar,9.1 kbar, 9.2 kbar, 9.3 kbar, 9.4 kbar, 9.5 kbar, 9.6 kbar, 9.7 kbar,9.8 kbar, 9.9 kbar, 10.0 kbar, 10.1 kbar, 10.2 kbar, 10.3 kbar, 10.4kbar, 10.5 kbar, 10.6 kbar, 10.7 kbar, 10.8 kbar, 10.9 kbar, 11.0 kbar,11.1 kbar, 11.2 kbar, 11.3 kbar, 11.4 kbar, 11.5 kbar, 11.6 kbar, 11.7kbar, 11.8 kbar, 11.9 kbar, and 12.0 kbar. “High pressure” for thepurpose of refolding steps, means about 0.25 to about 3.3 kbar.Specifically contemplated pressures within this range are 0.30 kbar,0.35 kbar, 0.40 kbar, 0.50 kbar, 0.55 kbar, 0.60 kbar, 0.65 kbar, 0.70kbar, 0.75 kbar, 0.80 kbar, 0.85 kbar, 0.90 kbar, 0.95 kbar, 1.0 kbar,1.1 kbar, 1.2 kbar, 1.3 kbar, 1.4 kbar, 1.5 kbar, 1.6 kbar, 1.7 kbar,1.8 kbar, 1.9 kbar, 2.0 kbar, 2.1 kbar, 2.2 kbar, 2.3 kbar, 2.4 kbar,2.5 kbar, 2.6 kbar, 2.7 kbar, 2.8 kbar, 2.9 kbar, 3.0 kbar, 3.1 kbar,3.2 kbar, 3.3 kbar.

A “host cell” is a microbial cell such as bacteria and yeast or othersuitable cell including animal or a plant cell which has beentransformed to express the heterologous protein of interest. Host cellswhich are contemplated by the present invention include those in whichthe heterologous protein expressed by the cell is sequestered inrefractile bodies. An exemplary host cell is E. coli K12, strain W311OG(pBGHI), which has been transformed to effect expression of the desiredheterologous protein.

“Native conformation” of a protein, in the present context, refers tothe secondary, tertiary and quaternary structures of a protein as itoccurs in nature in its fully active state.

“Pressurization” is a process of increasing the pressure (usually fromatmospheric or ambient presssure) to a higher pressure. Pressurizationtakes place over a predetermined period of time, ranging from 0.1 secondto 10 hours. Such times include 1 second, 2 seconds, 5 seconds, 10seconds, 20 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, 30minutes, 60 minutes, 2 hours, 3 hours, 4 hours, and 5 hours.

“Proteins” include a wide variety of peptide-containing molecules,including monomeric, dimeric, multimeric, heterodimeric, heterotrimeric,and heterotetrameric proteins; disulfide bonded protein; glycosylatedproteins; helical proteins; and α and β sheet-containing proteins.Particular proteins include hormones, antibodies, enzymes, and metalbinding proteins.

“Refolding,” “renaturing,” or “naturing,” in the present context, meansthe process by which a fully or partially denatured protein adoptssecondary, tertiary and quaternary structure like that of the cognatenative molecule. A properly refolded protein has biological activitythat is substantially that of the non-denatured molecule. Where thenative protein has disulfide bonds, oxidation to form nativeintramolecular disulfide bonds is a desired component of the refoldingprocess.

A “surfactant” is a surface active compound which reduces the surfacetension of water.

III. Reagents and Procedures

A. Chaotropic Agents

A chaotropic agent is a compound, including, without limitation,guanidine hydrochloride (guanidinium hydrochloride, GdmHCl), guanidinesulfate, urea, sodium thiocyanate, sarcosyl, sodium dodecyl sulfate,sodium octyl sulfate and/or other compounds which disrupt thenoncovalent intermolecular bonding within the protein, permitting thepolypeptide chain to assume a substantially random conformation.

Chaotropic agents, when employed, are used at “low” concentrations. Suchlow concentrations are 0 to about 4.5 M. Included are the particularconcentrations of 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.75M, 1.0M, 1.25M,1.5M, 1.75M, 2.0M, 2.25M, 2.5M, 3.0M, 3.5M, 4.0M, and 4.5M

B. Reducing and Oxidizing Agents

A reducing agent is capable of transferring electrons and, in so doing,“reducing” bonds between various atoms. In the context of the presentinvention, a reducing agent will disrupt intra- and intermolecularinteractions, in particular, those involving disulfide bridges.Exemplary reducing agents, according to the present invention, arediothiothreitol, glutathione, dithioerythritol, or β-mercaptoethanol.

Oxidizing agents can be used to neutralize reducing agents. Oxidizingagents include oxidized glutathione, molecular oxygen, air, or peroxides

C. Surfactants

Surfactants are used to improve the solubility of certain proteins.Useful surfactants include nonionic (including, but not limited to,t-octylphenoxypolyethoxy-ethanol and polyoxyethylene sorbitan), anionic(e.g., sodium dodecyl sulfate) and cationic (e.g., cetylpyridiniumchloride) and amphoteric agents. Suitable surfactants include, but arenot limited to deoxycholate, sodium octyl sulfate, sodium tetradecylsulfate, polyoxyethylene ethers, sodium cholate,octylthioglucopyranoside, n-octylglucopyranoside, alkyltrimethylammoniumbromides, alkyltrimethyl ammonium chlorides, and sodium bis(2-ethylhexyl)sulfosuccinate.

D. Buffering Agents

Buffering agents are advantageously present in disaggregating and/orrefolding mixtures to maintain a desired pH value or pH range. Inorganicbuffer systems (phosphate, carbonate, among others) and organic buffersystems (citrate, Tris, MOPS, MES, HEPES, among others) are well knownto the art.

E. Stabilizing Agents

Non-specific protein stabilizing agents act to favor the most compactconformation of a protein. Such agents include, but are not limited to,sucrose, trehalose, glycerol, betaine, amino acid(s), and trimethylamineoxide.

F. Spectroscopy

Another useful technique for optimizing refolding conditions is in situspectroscopic measurement of samples under pressure. This is awell-known process for examining protein stability under pressure, butit has been underutilized in protein aggregation studies. Using highpressure spectroscopic techniques to observe aggregates dissolve underpressure will help determine the optimal pressure ranges for recoveringproteins from aggregates. Custom made high pressure cells have beenroutinely used for high pressure unfolding studies and can be adaptedfor use in high pressure disaggregation and refolding.

IV. Current Methodologies for Protein Refolding

The following describe various generalized protein refoldingtechnologies representing the “state of the art”:

U.S. Pat. No. 5,077,392 (1991) describes a process for activation ofrecombinant protein produced in prokaryotes, in which the aggregatedproteins are dissolved in 4-8M guanidine hydrochloride or 6-10M urea.Once solubilized, the buffer is dialyzed to a pH between 1 and 4.Finally, the solution is diluted to provide a nondenaturing andoxidizing environment to allow for refolding.

U.S. Pat. No. 5,593,865 (1997) describes a process for activatingrecombinant disulfide bond-containing eukaryotic proteins afterexpression in prokaryote hosts. Inclusion body proteins are dissolved ina strong denaturing agent (6M guanidine hydrochloride) containingreducing agents. In the refolding step, proteins are introduced into anenvironment which is oxidizing and nondenaturing.

U.S. Pat. 4,677,196 (1987) also describes purification and production ofbiologically active proteins from insoluble inclusion bodies. This is ageneral method for recovering proteins from insoluble form includesdissolving the protein aggregates in SDS. Once dissolved, the proteinsolution is separated from SDS by column chromatography. In the absenceof SDS, the protein can refold. Finally, the protein is eluted from thecolumn. Urea has also been included in dissolved protein solutions.After anion exchange chromatography, the urea from the refolded proteinsolution is removed by dialysis.

U.S. Pat. No. 5,605,691 (1997) describes solubilization of inclusionbody proteins using SDS and heat. Once in solution, proteins arerefolded by first diluting the SDS and then dialyzing away the SDS tonondenaturing concentrations.

U.S. Pat. No. 4,659,568 (1997) describes a process for solubilization,purification and characterization of protein from insoluble proteinaggregates or complexes and compositions of matter therefrom. Theinsoluble protein aggregates or inclusion bodies are layered on top of aurea step gradient (3M to 7M urea). As the samples are centrifuged, theaggregates move through the gradient until they dissolve. This methodprovides a means of determining the urea concentration at which theprotein dissolves.

U.S. Pat. No. 5,728,804 (1995) describes a process in which denatured oraggregated proteins are suspended in a detergent-free aqueous mediumcontaining 5-7 M guanidine hydrochloride and incubated overnight. Oncesuspended, the sample is contacted with sufficient cyclodextrin toassist in the refolding of the proteins. Finally, the cyclodextrin isremoved by dialysis.

The following are patents disclosing processes developed for refoldingof particular proteins:

U.S. Pat. No. 4,652,630 (1987) describes a method for producing activesomatotropin. In this method, the aggregates or inclusion bodies aresolubilized in a chaotrope (3M to 5M urea), and the pH is adjusted toallow complete solubilization. Then the conditions are modified to allowoxidation in the presence of a nondenaturing concentration of chaotrope.

U.S. Pat. No. 5,064,943 (1991) also describes a method for solubilizingand renaturing somatotropin, but it does not require the use of achaotrope. Here, the pH is adjusted to between 11.5 and 12.5 andmaintained for 5 to 12 hours. Under these conditions, somatotropin willsolubilize and renature.

U.S. Pat. No. 5,023,323 (1991) describes a process for naturation ofsomatotropin (growth hormone) aggregates in which the aggregates aredissolved in a denaturing chaotrope (1M to 8M urea). The solubilizationstep is followed by exposing the sample to an oxidizing environment inthe presence of a nondenaturing concentration of chaotrope.

U.S. Pat. No. 5,109,117 (1992) describes a method in which somatotropinaggregates are dissolved in the presence of an organic alcohol andchaotrope (1M to 8M urea). Then the solubilized proteins are renaturedin a nondenaturing, oxidizing environment.

U.S. Pat. No. 5,714,371 (1998) provides a method for refoldingaggregates of hepatitis C virus protease. Aggregates are solubilized in5M guanidine hydrochloride. Second, a reducing agent is added to thesolution, and the pH is adjusted to provide an acidic pH. Third, thedenaturing agent is removed from the solution by dialysis, and finallythe pH is raised to its starting point.

U.S. Pat. No. 4,923,967 (1990) describes a process specific for humaninterleukin-2. Protein aggregates are dissolved in 4-8M guanidinehydrochloride with a sulfitolyzing agent. Once the proteins aredissolved, the sulfitolyzing agent is removed by solvent exchange.Finally, the temperature is raised to precipitate out interleukin-2 inpure form. To allow refolding, precipitates are dissolved again inguanidine hydrochloride plus a reducing agent. Finally, the solution isdiluted to refold proteins.

U.S. Pat. No. 5,162,507 (1992) describes a process for recoveringpurified, oxidized, renatured recombinant interleukin-2 frommicroorganisms. Insoluble interleukin-2 isolated from microorganisms issolubilized in 2M to 4M guanidine hydrochloride. The guanidinehydrochloride solution is then diluted until the proteins precipitateout of the solution. The precipitates are then redissolved in aguanidine hydrochloride solution. The proteins are then oxidized toreform native disulfide bonds. Finally, the solution is diluted andinterleukin-2 remains in solution.

U.S. Pat. No. 4,985,544 (1991) describes a process for renaturing fishgrowth hormone. In this process, the aggregates or inclusion bodies aredissolved using guanidine, urea, SDS, acid or alkali. The reducing agentis then removed, and an oxidizing agent is added. Finally, thedenaturing agent is removed to allow refolding.

U.S. Pat. No. 5,410,026 (1995) describes a method in which insoluble,misfolded insulin-like growth factor-1 (IGF-1) is refolded into anactive conformation. Once IGF-1 is isolated, it is incubated with 1-3Murea or 1M guanidine hydrochloride until the aggregates are solubilizedand refolded.

Other U.S. patents dealing with protein refolding include U.S. Pat. Nos.5,708,148; 4,929,700 and 4,766,224.

V. Agitation

Previous studies have demonstrated that agitation of proteins can resultin aggregation and precipitation Bam et al. (1998). However, the generaleffects of mass transfer have largely been ignored in protein refolding.The present inventors have found, surprisingly, that physical mixing or“agitation” (stirring, shaking, rotation, etc.) increases both the speedand extent of protein refolding.

Thus, according to the present invention, agitation of proteins can beemployed to assist or improve the refolding of proteins under pressure.Agitation can be applied to precipitated aggregates of proteinssuspended in an aqueous medium under high pressure. Optimally, suchagitation will be applied at intensities such that the precipitatedprotein aggregates are uniformly dispersed throughout the aqueoussolution, but below levels where agitation-induced aggregation isfavored. Such agitation should be applied as necessary to maintain thedispersion until such time as the protein disaggregates. Agitation canbe accomplished using ultrasound or by pumping through static mixingdevices.

VI. High Temperature Refolding

Although increased temperatures are often used to cause aggregation ofproteins, the present inventors have determined that increasedtemperatures can enhance refolding recoveries effected by high pressuretreatment, provided that the temperatures are not so high as to causeirreversible denaturation. Generally, the increased temperature forrefolding should be about 20° C. lower than the temperatures at whichirreversible loss of activity occurs. Relatively: high temperatures (forexample, about 60° C. to about 125° C., about 80° C. to about 110° C.,including about 100° C., about 105° C., about 110° C., about 115° C.,about 120° C. and about 125° C.) may be used while the solution is underpressure, as long as the temperature is reduced to a suitably lowtemperature before depressurizing, Such a suitably low temperature isdefined as one below which thermally-induced denaturation or aggregationoccurs at atmospheric conditions.

VII. Dialysis and Dilution Under Pressure

Protein refolding under pressure appears to be independent of proteinconcentration. This is surprising given that high protein concentrationis known to induce protein aggregation. However, while it is possible toobtain disaggregated, properly refolded protein at high concentrations,removal of the protein from pressure while still at high concentrationsmay result in re-aggregation. By the same token, running allpressurization steps under low concentration greatly increases thevolume of material that must be processed, and hence increases the timefactor. Alternatively, scaling up of the machinery to handle greatervolumes will increase speed. However, the cost for scaling thepressurized aspects of the machinery is prohibitive.

Thus, the present inventors propose to conduct disaggregation andrefolding experiments under high protein concentrations. “High”concentrations, depending on the protein in question, may range from 1mg/ml to 100 mg/ml. Following disaggregation, and either during or afterrefolding, the protein is diluted to a storage stable concentration of0.1 mg/ml to 10 mg/ml, preferably 1 mg/ml. This concentration isadjusted using various of the buffered solutions discussed above.

In addition, dialysis and dilution under pressure allow for changes inthe chemical environment surrounding proteins while under pressure, forexample, with reducing agents. Dialysis could be conducted in batchmode, where protein solutions are placed on one side of a dialysismembrane, and chemicals are allowed to diffuse across over time.However, proteins are too large to diffuse across the membranesutilized. In contrast, dilution would require an injection or mixing oftwo solutions while under pressure, creating a more sudden change inchemical conditions. In addition, one may wish to change pH of thesolutions being used in the refolding process. For example, rates ofdisulfide exchange in the presence of redox agents are typically higherat pH 10-11, while secondary structural stability of proteins istypically higher at lower pH's. Thus, in addition to permitting removalof redox reagents, dialysis under pressure allows a concomitant changein pH.

VIII. “Stepped” Depressurization

In another aspect of the present invention, applicants provide “stepped”depressurization. This process comprises dropping the pressure from thehighest pressure used to at least a secondary level that is intermediatebetween the highest level and ambient pressure. The goal is to providean incubation period at or about this intermediate pressure zone thatpermits a protein to adopt a desired conformation.

In one embodiment, the present invention merely contemplates anintermediate pressure incubation that lies somewhere between the highestand ambient pressures. Alternatively, there are particular pressureranges that may be suitable as “intermediate” pressure steps, includingbut not limited to about 50 MPa-500 MPa, about 100 MPa-400 MPa, andabout 200 MPa-300 MPa. Particular levels for intermediate pressurizationinclude about 100 MPa, about 150 MPa, about 200 MPa, about 250 MPa,about 300 MPa, about 350 MPa, about 400 MPa, about 450 MPa and about 500MPa. The only limitation is that, when used, the foregoing pressuresmust be lower than the first or highest level of pressurization. Timingfor intermediate depressurization steps include 1 min, 5 min, 10 min, 20min, 30 min, 45 min, 1 h, 2 h 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h,11 h and 12 h incubation periods.

IX. Batch versus Continuous Processing

Mechanically, there are two primary methods of high-pressure processing:batch and continuous. Batch processes simply involve filling a specifiedchamber, pressurizing the chamber for a period of time, anddepressurizing the batch. In contrast, continuous processes constantlyfeed aggregates into a pressure chamber and soluble, refolded proteinsmove out of the pressure chamber. In both set ups, good temperature andpressure control is essential, as fluctuations in these parameters cancause inconsistencies in yields. Both temperature and pressure should bemeasured inside the pressure chamber and properly controlled.

Batch Samples: There are many methods for handling batch samplesdepending upon the specific stability issues of each protein. Proteinsolutions can be loaded directly into a pressure chamber, in which caserefolding buffer would be used as the pressure medium. Alternately,samples can be loaded into any variety of sealed, flexible containers.This allows for greater flexibility in the pressure medium, as well asthe surfaces to which the protein is exposed. Sample vessels couldconceivably even act to protect the proteins of interest from chemicaldegradation (i.e., oxygen scavenging plastics are available).

Continuous Processing: With continuous processing, scale-up is simple.Small volumes under pressure can be used to refold large volumes ofprotein solutions. In addition, using an appropriate filter on theoutlet of a continuous process will selectively release properlyrefolded proteins from the chamber while retaining both soluble andinsoluble aggregates.

X. Multimeric Proteins

Many proteins that have potential uses in human disease therapies,prophylactic applications, and diagnostic applications are composed ofmultiple protein chains. Production of these proteins by recombinanttechniques requires that the appropriate chains assemble to form thenative secondary, tertiary, and quaternary structure. Improperlyassociated or unassociated chains are particularly prone to formnon-native aggregates and precipitates. In one example, these non-nativeaggregates can be suspended in a suitable formulation in a samplecontainer, placed in a commercially available high-pressure vessel suchas those commercially available from the Flow International Corp. orHigh Pressure Equipment Co., and pressurized to disaggregate the proteinand promote folding and assembly to the native structure. In anotherexample, individual chains may be synthesized separately throughwell-known recombinant techniques. Purified or semi-purified solutionsof individual chains may be combined in a sample holder in a suitableformulation, and placed in the aforementioned high-pressure vessel, andpressurized to effect folding and assembly to the native multimericprotein.

In particular embodiments, the invention provides for the refolding ofmultimeric proteins, such as dimers, trimers, tetramers, pentamers,hexamers, septamers, octamers, nonamer, etc. The polypeptide subunitscan be identical (homodimer, etc.) or one or more can differ within thenative protein (heterodimers, etc.). In a specific example discussedbelow, the protein is recombinantly produced human interferon-γ, whichis a homodimer.

A. Interferon

rhIFN-γ forms aggregates of completely different morphology when exposedto temperatures just below the onset of the thermal transition ascompared to aggregates formed by exposure to mild concentrations ofguanidine hydrochloride. Thermally-induced aggregates of rhIFN-γ arefibrous structure, whereas guanidine hydrochloride-induced aggregatesform an amorphous precipitate. The secondary and tertiary structures ofthe aggregates, which are distinct from the native state, areindependent of aggregate morphology.

Pressure is effective in the dissolution of both fibrous and amorphousaggregates of rhIFN-γ and in the recovery of the native structure, asmeasured by second derivative UV and FTIR. The rate of dissolution ofthe amorphous aggregates is rapid and acquisition of thepressure-dissociated state is achieved within 30 minutes at 250 MPa. Theacquisition of the pressure-dissociated state from thermally-inducedaggregates is approximately four times slower than that for theamorphous aggregates. However, once the pressure-dissociated state isachieved, the rate and extent of recovery of the native state (i.e.,rate of refolding and yield), as measured by second derivative UV, isindependent of the initial aggregate form.

The extent of refolding at 100 MPa for 1.5 hours was dependent on theprotein concentration during refolding, with recovery of native-likestructure increasing with decreasing protein concentration. For samplesdiluted to 1 mg/mL after the refolding protocol, the extent of refoldingwas independent of protein concentration during refolding. Thedifferences in protein concentration dependence are attributed to asignificant population of monomer remaining in the high proteinconcentration samples upon depressurization. Prompt dilution of the highprotein concentration samples to 1 mg/mL following the refoldingprotocol results in conditions favoring refolding over aggregation,which proceeds via the monomer. As such, acquisition of dimer isessentially complete and is not affected by protein concentration duringrefolding. Failure to dilute the higher protein concentration samplesafter the pressure treatment results in conditions that favoraggregation over refolding and the high concentration of monomer leadsto significant aggregation.

The equilibrium reaction of rhIFN-γ by pressure up to 250 MPa may befollowed by second derivative UV spectroscopy. This equilibrium reactionis concentration dependent and is thus dissociation of the native dimer.The dissociation has a ΔV equal to −209+/−13 mL/mol of dimer, which isindependent of protein and sucrose concentrations. Sucrose stabilizesrhIFN-γ against dissociation, via preferential exclusion at the proteinsurface, by shifting the equilibrium toward the more compact nativestate. The surface area change of dissociation was measured to be12.7+/−1.6 nm2/molecule of dimer, which represents a ca. 30% increase inthe solvent-exposed surface area over the native dimer. A comprehensiveequation was developed from experimental data that predicts theΔG_(diss) of rhIFN-γ as a function of pressure, sucrose and proteinconcentrations.

Using similar criteria as the equilibrium dissociation, secondderivative UV spectroscopy may be used to follow the rate of aggregationof rhIFN-γ. However, because similar criteria are used, solutionconditions must be chosen so that significant dissociation does notoccur coincident with aggregation. The inventors have shown that theE_(a) for the first-order guanidine hydrochloride-induced aggregationwas measured to be 130+/−30 kJ/mol dimer, which is significantlydifferent from the E_(a) of the second-order thermally-inducedaggregation process. The disparity in the activation energies for theaggregation reactions induced by the different stresses is confirmationthat the kinetics of aggregation for the guanidine hydrochloride-inducedaggregation are dominated by the N to 2M dissociation.

As was previously reported (Kendrick et al., 1998a), sucrose reduces theaggregation rate of rhIFN-γ in the presence of guanidine hydrochlorideby preferential exclusion of sucrose from the protein surface, shiftingthe equilibrium toward the compact native state, N, and away from theexpanded transition state, N*. However, pressure destabilizes rhIFN-γ toaggregation by guanidine hydrochloride through increased solvation ofthe protein. The increased solvation increases the exposed surface areaof the protein, shifting the equilibrium away from N and toward N* and,in this way, counteracts the stabilizing effect of sucrose. The changein exposed surface area and change in partial molar volume between theexpanded N* and N were found to be 3.5+/−0.2 nm²/molecule and −39+/−9mL/mol of dimer, respectively. Thus, the perturbation required for theformation of the transition-state species, which leads to the formationof the aggregate competent monomer, is small in comparison to thedifference between the native and dissociated states.

B. Other Multimers

Examples of other multimeric proteins which may be refolded according tothe present invention include hemoglobin, lactic acid dehydrogenase,antibodies and antibody. fragments.

XI. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 High Pressure Disaggregation and Refolding

Procedure 1: High Pressure Protein Disaggregation and Refolding VaryingTemperature

Protein aggregation: Recombinant human growth hormone (rhGH) wassuspended in 10 mM sodium citrate buffer (pH 6.0, 1 mM EDTA, 0.1% sodiumazide) at a concentration of 1.5 mg/ml. 10 mL of rhGH solution wastumbled end over end in a 15 ml conical falcon tube at 8 rpm for atleast 24 hours. Aggregates showing greater structural damage weretumbled under identical conditions with the addition of 0.75M guanidinehydrochloride in the citrate buffer.

Sample Preparation: One-half the required volume was centrifuged at13,000 g for 15 minutes to sediment the insoluble aggregates.Supernatant was decanted and replaced with the final volume of buffer(10 mM sodium citrate buffer, pH 6.0, 1 mM EDTA, 0.1% sodium azide). Theaggregate pellet was resuspended with an Ultrasonics brand sonicator(50% duty, 1 second pulse cycle). Once resuspended, samples were placedinto 1 ml syringes (end heat-sealed and plunger in place to remove allheadspace).

Pressurization and Analysis: After temperature had equilibrated, sampleswere placed into the pressure chamber. Pressure was increased to 2 kbarfor 24 hours. Atmospheric samples were placed at an identicaltemperature. Pressure was released gently and evenly over 15 minutes.After depressurization, samples were centrifuged at 13,000 g for 15minutes. Supernatant was analyzed for soluble protein by absorbance at278 nm. Light scattering components were subtracted utilizing the methoddescribed by Leach & Scheraga (1960). The extinction coefficient forrhGH is 18,890 (cm mol/l)⁻¹.

Results of High pressure protein disaggregation and refolding varyingtemperature: In the absence of guanidinium, yields of refolded humangrowth hormone after pressure treatment increase with increasingtemperature, reaching approximately 100% at a temperature of 60° C.Exposure to similar temperatures without high pressure treatment did notresult in significant improvements in yields of folded, native proteins.

Procedure 2: Refolding Aggregates Using High Pressure Dialysis

Protein Aggregation and Sample Preparation: Hen egg white lysozyme (40mg/ml) from Sigma Chemical Co. was denatured and reduced in 8M guanidinehydrochloride (GdmHCl), 40 mM DTT for one hour. The protein solution wasthen shock-diluted 20-fold with 50 mM Tris-HCl buffer (pH 8.0, 1 mMEDTA) to induce aggregation. Samples were then diluted an additional2-fold with Tris-HCl buffer (pH 8.0+GdmHCl+DTT) to create the finalrefolding solution to be placed inside the dialysis tubing (1 mg/mLlysozyme in 50 mM Tris-HCl pH 8.0, 0.8M GdmHCl, 100 mM DTT). Sample wasthen injected into dialysis tubing (3500 molecular weight cutoff, soakedovernight in Tris-HCl buffer, pH 8.0). Just prior to pressurization, thedialysis tubing was placed in a 25-fold volume excess of Tris-HCl buffer(pH 8.0, 0.8M GdmHCl, 3 mM oxidized glutathione) and sealed forpressurization.

Pressurization and Analysis: As quickly as possible following insertionof the dialysis tubing into the exchange buffer, samples werepressurized to 2 kbar. Pressure was maintained for 5 days. Samples weredepressurized gently over 15 minutes. Once at atmospheric pressure,samples were centrifuged for 15 minutes at 13,000 g. to sedimentinsoluble aggregates. The supernatant was analyzed for enzymaticactivity as previously described (St. John et al., 1999).

Example 2 High Pressure Refolding of Fibrous and Amporphous Aggregatesof rhIFN-γ Materials and Methods

Purified recombinant DNA derived rhIFN-γ in 5 mM sodium succinate, pH5.0 (succinate buffer) was provided by Genentech Inc., stored at 4° C.until use and used without further purification. 40 mM pyridine acetate,pH 5.0 buffer (PyrAc buffer) was used for gas-phase electrophoreticmobility mass analysis (GEMMA). Succinate buffer was used in all otherexperiments. Protein standards (bovine serum albumin, glucose oxidase,hemoglobin, ubiquitin and thyroglobulin) were purchased from Sigma. Allchemicals were of reagent grade or higher and were purchased from Sigmaas well.

Aggregate preparation. Aggregates were prepared by inducing aggregationeither by raising the solution temperature to 40° C. or by addingguanidine hydrochloride to the protein solution such that the finalguanidine hydrochloride concentration was 0.45 M. The extents of theaggregation reactions were in excess of 95% in all cases. The inventorshave determined the extent of reactions from the well-characterizedaggregation rates both for the thermally-induced aggregation, and foundsecond-order in protein concentration; for the guanidinehydrochloride-induced aggregation reaction, first-order in proteinconcentration. Guanidine hydrochloride-induced aggregates were washedwith sufficient fresh buffer so that the final guanidine hydrochloridesolution concentration was below 5 mM. Aggregates for refoldingexperiments were suspended in fresh buffer at approximate concentrationsof 1, 10 and 20 mg/mL rhIFN-γ.

High-pressure experiments. The high-pressure equipment used forrefolding experiments consisted of a high-pressure reactor,high-pressure UV spectroscopy cell, metallic gauge (accurate to +/−2MPa) and generator. The high-pressure reactor and high-pressure UVspectroscopy cell were designed and fabricated in the inventors'laboratory. The gauge and generator were purchased from High PressureEquipment, Co, (Erie, Pa.). For any given experiment, either thehigh-pressure reactor or high-pressure UV spectroscopy cell wasemployed.

The high-pressure UV spectroscopy cell was made of 316 stainless steel,sealed with Buna-N 90 durometer o-rings and had an optical port diameterof 6 mm and pathlength of 7.65 mm. The cell utilized cylindricalsapphire windows (16 mm diameter, 5.1 mm thick) and was capable ofexperiments up to 250 MPa. Separation of the sample from the pressuretransmitting fluid (silicon oil) was facilitated by a piston deviceexternal to the cell. All wetted metal surfaces were constructed of 316SS. All in situ high-pressure UV experiments were conducted at a proteinconcentration of ca. 1 mg/mL.

For experiments conducted in the high-pressure reactor, aggregatesuspensions were loaded into ca. 1 mL polypropylene bulbs, heat-sealedand loaded into the reactor. The reactor was sealed and the pressureincreased to 250 MPa, which is sufficient pressure to ensure that, atequilibrium, rhIFN-γ is fully dissociated into monomers, as determinedby the inventors. The samples were held at 250 MPa for sufficient timeto dissolve the aggregates and for the protein equilibrate, based uponin situ second-derivative UV measurements (see below). The pressure wasthen lowered to 100 MPa and again held for sufficient time to reachequilibrium for rhIFN-γ concentrations of ca. 1 mg/mL (see below), afterwhich the pressure was reduced to 0.1 MPa and the samples were removedfrom the high-pressure reactor.

Transmission Electron Microscopy (TEM). TEM was performed on samples ofnative, aggregated and pressure-treated aggregates rhIFN-γ that had beennegatively stained with a 2% solution of uranyl acetate at total aprotein concentration of ca. 1 mg/mL. Specimens were stained onformvar/carbon 400 mesh copper grid and were viewed on a JOEL 100CX TEMwith an accelerating voltage of 80,000 kV. Micrographs of aggregatestructures were obtained at magnifications from 20,000 to 80,000.

Gas-phase electrophoretic mobility mass analysis (GEMMA). The procedurefor GEMMA particle size analysis was based on the method of Kaufman etal. (1996) (Kaufman et al., 1996). The instrument was calibrated usingprotein standards of known molecular weight (MW). A linear calibrationcurve was developed from the natural log of the electrophoretic mobility(EM) diameters versus the natural log of MW. Samples of native,aggregated and pressure-treated aggregates of rhIFN-γ were diluted toca. 1 to 2 μg/mL into PyrAc buffer, which has a conductivity of ca. 2mΩ⁻¹ cm, and immediately analyzed using a GEMMA analyzer from TSI (St.Paul, Minn.). PyrAc buffer was employed due to the GEMMA requirement ofless than 1 ppm non-volatile material in the sample.

The GEMMA analyzer consisted of a model 3480 electrospray aerosolgenerator, a model 3085 differential mobility analyzer (DMA), a model3025A ultrafine condensation particle counter (CPC). Dried and filteredair was delivered at a rate of 1 Lpm from the utility air supply througha model 307402 filter/dryer and instrument-grade carbon dioxide wasdelivered at a rate of 0.05 to 0.1 Lpm. The fused-silica electrospraycapillary was 25 cm long and had an internal diameter of 25 μm. Sampleflow (ca. 100 nL/min) was facilitated by a differential pressure of 25kPa (3.7 psi) across the capillary. An electrical potential of 2 kV wasmaintained across the capillary, with a corresponding current of 200 to250 nA.

Data acquisition was made via Aerosol Instrument Manager (AIM©) versionβ4.10 software from TSI on an IBM compatible PC running MicrosoftWindows 95©. The data acquisition software used 64 log-spaced channelsper decade of EM diameter. Sample analysis was made from ca. 2.5 to 58nm and acquisition times from 3 to 6 minutes, using assumed values forair viscosity (μ) of 1.82×10⁻⁵ kg/(m s), mean free path (λ) of 7.75×10⁻⁸m and protein density (ρ) of 1.2 g/mL. Data files, in units of masspercent concentration, were exported as text files and imported intoMicrosoft Excel©. EM diameters were determined by the maximum of a givenpeak. Monomer to dimer ratios and extent of reactions (based on masspercent dimer) were determined by finite integration of the individualpeaks in the mass percent distributions.

Dynamic light scattering (DLS). Dynamic light scattering measurementswere performed on the thermally-induced aggregates of rhIFN-γ using aNicomp 370 Submicron Particle Sizer (Particle Sizing Systems, SantaBarbara, Calif.). The total protein concentration was 1 mg/mL.

Derivative UV spectroscopy. Absorption spectra were measured with aPerkin-Elmer Lambda 3B dual-beam spectrophotometer at rhIFN-γconcentrations of ca. 1 mg/mL. Scans were measured from 310 nm to 250 nmwith scan rate of 15 nm per minute. Data acquisition was made via aNational Instruments (Austin, Tex.) model AT-MIO-16E-10 data acquisitionboard at a rate of 5 samples per second. National Instruments LabView©software was used to control data acquisition and Microsoft Excel© toconvert the wavelength and absorption data from volts to nm andabsorbance units, respectively. The second derivatives of the absorptionspectra (d²A/dλ²) were calculated in Grams/386 (v. 3.02) software(Galactic Industries) using the Savitzky-Golay method with a secondorder polynomial smoothed over +/−2 nm.

Temperature was controlled using recirculating fluid thermostated by aLauda model M3 recirculating bath temperature controller. Spectra forsamples treated in the high-pressure reactor were collected atatmospheric pressure with the samples in standard 10 mm pathlengthquartz cuvettes and the appropriate blank placed in the reference cellof the spectrophotometer. For experiments carried out in thehigh-pressure cell, absorption spectra were collected for the sample andbuffer separately, with no reference sample in the spectrophotometer.Subtraction of the buffer spectrum, collected at the appropriatepressure, from the protein spectrum was carried out in Grams/386 (v.3.02) software prior to calculation of the second derivative.

Fourier Transform IR Spectroscopy (FTIR). IR spectra were collectedusing an adjustable pathlength cell set at 8 μm. Blank buffer spectrawere collected using the same cell under an identical solutioncondition, with no protein present. All spectra were collected atatmospheric pressure on a Nicolet Magna-IR© 750 series II (Madison,Wis.) spectrometer equipped with a DTGS detector. Interferograms werecollected in the single beam mode, signal-averaged over 256 (for samplesat ca. 20 mg/mL) and 1024 (for samples at ca. 10 mg/mL) scans at aresolution of 4 cm⁻¹ using Omnic© (v. 2.1) Software from Nicolet. Theoptical bench and sample chamber were continuously purged with dry airsupplied from a Whatman model 75-52 FTIR purge gas generator (Haverhill,Mass.). Single beam spectra of both protein-in-buffer and buffer werereprocessed into absorbance spectra by subtracting out the backgroundspectra from each. Nicolet software was used to subtract the buffer andwater vapor contributions from the protein-in-buffer spectra and tocalculate the second derivative. The remaining spectrum was 7-pointsmoothed to remove white noise and imported into Grams/386© (v. 3.02)software (Galactic Industries) where it was baseline corrected and areanormalized per method of Dong et al., 1995.

Size Exclusion Chromatography (SEC). The presence of soluble aggregatesand monomer/dimer content of pressure-treated aggregates was measured bySEC and compared to the liquid control. Insoluble aggregates wereremoved by centrifugation and the supernatant was assayed by sizeexclusion chromatography (SEC). 50 μL aliquots of the supernatant,diluted to 1 mg/mL in succinate buffer, were loaded onto a silica-basedTosohaas TSK-GEL G2000SW_(XL) column. The mobile phase, 1.2 M KCl, waspumped at a rate of 0.8 mL/min and absorbance at 214 nm was monitored asa function of time. Resultant chromatograms were imported intoGrams/386© (v. 3.02) software where they were area normalized and curvefit to determine the percent contributions and elution times for themonomer and dimer. The Autofind curve fit function was employed withparameters set for a maximum of two peaks, Guassian curve, mediumsensitivity and offset baseline. The instrument was calibrated usingprotein standards of known MW. A linear calibration curve was developedfrom the natural log of the elution time versus the natural log of MW.

Results and Discussion

Characterization of aggregates: Physical characterization. At proteinconcentrations above 5 mg/mL, thermally-induced aggregates of rhIFN-γformed a colorless, transparent gel with high viscosity that could notbe concentrated by centrifugation. At concentrations of ca. 1 mg/mL, theprotein concentration was sufficiently low that the viscosity increaseupon aggregation was minimal and separation by centrifugation waspossible. Like the aggregates formed at higher protein concentrations,the aggregates formed at concentrations of ca. 1 mg/mL were colorlessand scattered relatively little light. In contrast to aggregates formedby high temperatures, the addition of guanidine hydrochloride tosolutions of rhIFN-γ resulted in the formation of a white, opaqueprecipitate that was easily centrifuged from solution, regardless of theaggregation protein concentration.

Electron micrographs of the aggregates revealed that thethermally-induced aggregates formed a matrix of fiber-like strands ofindeterminate length (FIG. 2A), but the guanidine hydrochloride-inducedaggregates formed an amorphous precipitate (FIG. 2B). Thethermally-induced aggregate fibrils have a consistent diameter on theorder of 10 nm. No amorphous structures were observed in thethermally-induced aggregates. Conversely, no fibrous structures werefound in the guanidine hydrochloride-induced aggregate samples.

The EM diameter distribution of native rhIFN-γ and thermally-inducedaggregates was measured by GEMMA and the results are presented in FIG.3. The native protein, equilibrated at 25° C. and 1 mg/mL in PyrAcbuffer, was found to have two significant peaks centered at 4.4 and 5.5nm (FIG. 3A). The peaks corresponded to particles having molecularweights of 16 and 32 kD, respectively, based on the calibration curvewith known protein standards. These molecular weights are in goodagreement with the known molecular weights for the monomer and dimer ofrhIFN-γ (16.45 and 32.9 kD, respectively). No other significant peakswere detected in the native sample if the concentration of the samplewas kept below ca. 3 μg/mL. The mass percent monomer detected by GEMMA(ca. 12% in FIG. 3A) has been determined to be artificially high fromdissociation equilibrium experiments. The bias towards the monomer iscaused by surface-induced dissociation of the native dimer on theelectrophoresis capillary. Corrected mass percent monomer for nativerhIFN-γ, and EM diameters and effective molecular weights for themonomer and dimer, as determined by GEMMA, are reported in Table 1A.

TABLE 1A Comparison of GEMMA results between the native control andpressure-treated aggregates of rhIFN-γ. Native control Pressure-treatedaggregate Mass percent monomer 1.4 +/− 3.5%¹ 12 +/− 3.5%¹ Monomer EMdiameter 4.4 nm 4.6 nm Dimer EM diameter 5.5 nm 5.7 nm Monomer effectiveMW³ 16 kD 18 kD Dimer effective MW³ 32 kD 36 kD

TABLE 1B Comparison of SEC results between the native control andpressure- treated aggregates of rhIFN-γ. Native control Pressure-treatedaggregate Mass percent monomer²    6.4 +/− 1.5%    4.2 +/− 1.5% Monomerelution time 889 +/− 1 s   889 +/− 1 s   Dimer elution time 830 +/− 1s   821 +/− 1 s   Monomer effective MW⁴ 16 kD 16 kD Dimer effective MW⁴33 kD 35 kD *There was no observed effect with aggregate type orrefolding concentration. ¹Corrected mass percent monomer calculated fromthe measured equilibrium constant. ²Calculated by Guassian curve-fitusing Grams/386 © (v. 3.02) as described in text. ³Calibration curvepresented elsewhere. ⁴Calibration curve not shown.

The thermally-induced aggregate sample, aggregated at 40° C. and 21mg/mL for 48 hours in succinate buffer, had ca. 1 and 4% by mass ofmonomer and dimer, respectively. The aggregates (FIG. 3B), in excess of95% by mass, had a mass average diameter of 16.5 nm, corresponding to anaverage effective MW of 980 kD based on the calibration curve with knownprotein standards. The effective diameter from GEMMA agreed poorly withDLS results, which indicated an effective hydrodynamic diameter of 1900nm, and the fact that the aggregates were visible to the eye. It islikely that, as droplets are formed at the exit of the capillary, thefibrous aggregates are sheared into smaller aggregates (Kaufman, 2000).Thus, it was determined that GEMMA is not an appropriate technique forassessing the size of the aggregates, but is appropriate for detectingthe presence of aggregates and determining the mass percent aggregate.The particle diameter obtained from DLS measurements was consideredinaccurate due to the morphology of the thermally-induced aggregates andwas used only for gross comparison with the GEMMA result. Guanidinehydrochloride-induced aggregates were not analyzed by GEMMA due to therequirement of less than 1 ppm non-volatile material in the sample.

Characterization of aggregates: Structural characterization. The regionbetween 275 and 295 nm of the second derivative UV spectrum reflects themicroenvironments of tryptophan and tyrosine residues and is affected bythe conformational state of proteins (Balestrieri et al., 1978; Ragoneet al., 1984; Servillo et al., 1982). Since the derivative UV spectra oftyrosine and tryptophan are minimally affected by pressure (Lange etal., 1996), changes to the second derivative UV spectrum of rhIFN-γ bypressure result from changes to the native conformation, as shown by theinventors here. The second derivative UV spectra of native, pressuredissociated soluble protein and thermally-induced and guanidinehydrochloride-induced aggregate forms of rhIFN-γ in 5 mM sodiumsuccinate buffer are presented in FIG. 4. The wavelength position of theextremum located near 286 nm for native and dissociated forms of rhIFN-γis independent of the degree of dissociation and its relative height hasbeen shown by the inventors to indicate the degree of dissociation ofrhIFN-γ. Thus, the relative height of this extremum can also be used tofollow the extent of reassociation and refolding of rhIFN-γ in theabsence of aggregates.

The spectra of the two aggregate forms (thermal- and guanidinehydrochloride-induced) show similar deviations from the native secondderivative UV spectrum and are distinct from the both the native andpressure-dissociated spectra (FIG. 4). The aggregate spectra display anoverall dampening of the signal amplitude and a blue-shift in theabsorbance spectra, both of which indicate increased exposure tohydrophilic environments of the tryptophan and tyrosine residuesrelative to the native structure (Lange et al., 1996; Mach & Middaugh,1994). Additionally, both aggregate spectra show significant reductionin the depth of the minimum located near 280 nm. However, there arenotable differences between the aggregate second derivative UV spectra.The extent of perturbation from the native spectrum, as measured byamplitude reduction and wavelength shift, is less severe with thethermally-induced aggregate spectrum compared with the guanidinehydrochloride-induced aggregate spectrum. Further, there is significantband broadening in the maxima located near 288 and 280 nm for theguanidine hydrochloride-induced aggregate spectrum that is not apparentin the thermally-induced aggregate spectrum.

To compare the secondary structures of the two aggregates with eachother as well as the native form, FTIR absorbance spectra of all threestates were collected and second derivative spectra calculated andcompared. FIG. 5 is a plot of the second derivative FTIR spectra ofnative rhIFN-γ in buffer and both the thermal- and guanidinehydrochloride-induced aggregates of rhIFN-γ. Significant perturbationsfrom the native state are observed in both aggregate forms, with awavelength shift of and a loss of absorbance in the α-helix band (near1656 cm⁻¹ in the native spectrum) and concomitant appearance ofintermolecular β-sheet bands near 1620 and 1695 cm⁻¹ (FIG. 5). Thecontributions of the intermolecular β-sheet bands near 1620 and 1695cm⁻¹ are comparable for both aggregate forms, as is the extent of lossin α-helix. Only minor differences in β-sheet (region near 1630 to 1645cm⁻¹) and turn structures (region near 1670 to 1685 cm⁻¹) are apparentbetween the temperature- and guanidine hydrochloride-induced aggregates.

High pressure dissolution and refolding kinetics. Pressure is known todisfavor the higher-order association of protein subunits (Silva & Weber1993) and therefore may be employed to dissociate aggregates, as hasbeen previously demonstrated (Foguel et al., 1999; Gorovits & Horowitz1998; Silva et al., 1989; St. John et al., 1999). When guanidinehydrochloride-induced aggregates of rhIFN-γ were exposed to 250 MPa,there was rapid and complete loss of light-scattering aggregates, asmeasured by absorbance at 310 nm (FIG. 6A). The total rhIFN-γconcentration was 1 mg/mL and the guanidine hydrochloride concentrationin solution was ca. 5 mM. Once dissolution of the aggregates wascomplete, absorbance scans were collected at 250 MPa and derivativespectra calculated. The pressure was maintained at 250 MPa until thesecond derivative spectrum no longer changed with time (ca. 30 minutes).The inventors found that second derivative spectrum of guanidinehydrochloride-induced aggregates at 250 MPa equilibrated to the spectrumobtained when native rhIFN-γ is dissociated with pressure at 250 MPa(FIG. 4), indicating complete disruption of the aggregate structure anddissociation of the dimeric structure to monomer.

Once the second derivative spectrum displayed no further changes withtime at 250 MPa, the pressure was reduced to 100 MPa and absorbancespectra collected over time. The second derivative spectra were againcalculated and compared until changes with time were no longer observed.At 100 MPa, the extremum near 286 nm (a maximum in the pressuredissociated spectrum, see FIG. 4) decreased in height with time andeventually became a minimum as the protein began to assume a more nativeconformation. FIG. 6B is a plot of the height of the extremum near 286nm versus time at 100 MPa for the reassociation/refolding of rhIFN-γ at1 mg/mL, after the guanidine hydrochloride-induced aggregates weredissociated at 250 MPa. Superimposed on FIG. 6B are heights of thespectra for the pressure dissociated and native forms of rhIFN-γ as wellfor the equilibrium pressure-refolded form (1 mg/mL rhIFN-γ), achievedby lowering the pressure to 0.1 MPa. The refolding at 100 MPa iscomplete in ca. one hour and the 286 nm extremum height of thepressure-treated aggregate has returned nearly completely to that of thenative control.

The rate of dissolution of thermally-induced aggregates at pressurecould not be quantified by absorbance changes at 310 nm with timebecause the aggregates did not scatter light sufficiently. As well, thedissolution of aggregates at pressure can not be followed in the regionbetween 275 and 295 nm either because aggregate dissolution andstructural migration to the pressure-dissociated form (FIG. 4) occursimultaneously. Thus, the rate of thermally-induced aggregatedissolution at pressure cannot be directly compared with the pressuredissolution of guanidine hydrochloride-induced aggregates. However, thetime required for the thermally-induced aggregate to assume thepressure-dissociated state (ca. 2 hours), measured by second derivativeUV, is significantly slower compared to the time required for guanidinehydrochloride-induced aggregates (ca. 30 minutes) to assume thepressure-dissociated state (data not shown). But once thepressure-dissociated state is achieved, the refolding rates arecomparable (FIG. 6B).

Characterization of pressure-treated aggregates. Thermally- andguanidine hydrochloride-induced aggregates of rhIFN-γ in buffer atprotein concentrations of ca. 1, 10 and 20 mg/mL were pressurized to 250MPa for five hours. The pressure was then lowered to 100 MPa for one andone-half hours, then again lowered to 0.1 MPa. Analysis of the pressuretreated aggregates was made both immediately following and two weeksafter pressure treatment to assess the effectiveness of the pressuretreatment on acquisition of native-like characteristics from aggregatesand the stability of the pressure-treated aggregates againstre-aggregation, respectively. TEM, GEMMA and HPLC were employed tophysically characterize the pressure-treated aggregates and secondderivative UV and FTIR spectroscopies were used to structurallycharacterize the pressure-treated aggregates. Comparison of the resultsfor each technique was made among the pressure-treated aggregates, theaggregate controls and native liquid rhIFN-γ control.

Physical characterization after pressure treatment. Upondepressurization, all thermally- and guanidine hydrochloride-inducedaggregate samples were optically clear with no particulates visible tothe eye. Additionally, the 10 and 20 mg/mL thermally-induced aggregatesamples, which were gelatinous solids prior to pressure treatment, wereliquids with viscosities indistinguishable (judged qualitatively) fromthe native liquid controls.

TEM was performed on the liquid control and on the 1 and 20 mg/mL(pressurized and control) samples for both types of aggregates at atotal protein concentration of ca. 1 mg/mL (20 mg/mL samples werediluted immediately after pressure treatment). FIG. 7 containsrepresentative TEM micrographs of pressure-treated (A) thermally-inducedand (B) guanidine hydrochloride-induced aggregates of rhIFN-γ along with(C) the liquid control. Aggregate controls were unchanged from thestructures initially observed and, therefore, micrographs of theaggregate controls are not presented in FIG. 7 (see FIG. 2 forrepresentative structures of the aggregate controls). All micrographs inFIG. 7 are at a magnification of 40,000. In both pressure-treatedsamples and the liquid control, amorphous and fibrous structures wereobserved, with amorphous material more prevalent than fibrous. FIG. 7A2displays an amorphous structure from pressure-treated thermally-inducedaggregates, which is representative of the dominant structure found forall three samples of rhIFN-γ. The observed structures and frequency ofamorphous material relative to fibrous material in both pressure-treatedaggregates and the liquid control were consistent. Additionally, therewas no observed difference between the samples that werepressure-treated at 1 or 20 mg/mL for both the thermal- and guanidinehydrochloride-induced aggregates (comparison not shown). The fibrousnetwork observed in the thermally-induced aggregate samples prior topressure treatment was destroyed and a large proportion of thepressure-treated thermally-induced aggregate was amorphous. As well, thefibrous material that was observed in the pressure-treated guanidinehydrochloride-induced sample was not found in the guanidinehydrochloride-induced aggregate controls.

The fibrous structures seen for the pressure-treated aggregates and theliquid control were shorter in length and more varied in diameter thanthe fibrous structures observed in the thermally-induced aggregatecontrols. The lengths of the fibrous structures in the pressure-treatedaggregates and the liquid control were generally 100 to 1000 nm inlength and the diameters were typically ca. 12 nm but ranged up to ca.25 nm. Some intertwined networks of fibrils were observed in all threesample sets and examples are given in FIGS. 7A3 and 7C2. It wasconcluded that the morphologies of the pressure-treated aggregates areindistinct from the control and that TEM sample preparation, whichinvolves drying of the protein sample, induced structures consistentwith both amorphous and fibrous aggregates.

Structural characterization after pressure treatment. Following thepressure-treatment, UV and FTIR absorbance spectra were collected andsecond derivative UV and FTIR spectra calculated for pressurized andcontrol samples. The 10 and 20 mg/mL samples were diluted immediatelyafter depressurization such that all UV analysis was conducted at 1mg/mL rhIFN-γ. Due to concentration limitations of the technique, FTIRspectroscopy was performed only on the 10 and 20 mg/mL samples.

FIG. 8 is a plot of the second derivative UV spectra of (A)thermally-induced and (B) guanidine hydrochloride-induced,pressure-treated aggregates. Spectra were recorded at 0.1 MPa after thepressure-treatment protocol. For reference, the second derivative UVspectrum of native rhIFN-γ is superimposed on both windows of FIG. 8.The aggregate control spectra are omitted from FIG. 8 for clarity (seeFIG. 4 for spectra of aggregates). The extent of recovery of the nativesecond derivative UV spectrum is aggregate-form independent. But, thedegree of recovery of the native second derivative UV spectrum isconcentration dependent, with greater recovery of the native spectrum atlower protein concentrations. At 1 mg/mL, the pressure-treatedaggregates recover nearly identical second derivative UV spectra as thenative rhIFN-γ liquid control. Table 2 contains the summary of thepercent recovery of the native spectrum measured by second derivative UVspectroscopy, using the ratio of the difference in heights of theextrema near 286 nm between the refolded and pressure-dissociated staterelative to those of the native and pressure-dissociated states.

TABLE 2 Recovery of native spectral features for pressure-refoldedaggregates of rhIFN-γ measured by second derivative UV and FTIR.Guanidine Thermally-induced hydrochloride-induced Conc. Percent Percentduring recovery of Area of recovery of Area of Refolding Extremum nearOverlap of Extremum near Overlap of (mg/mL) 286 nm UV FTIR spectra 286nm UV FTIR spectra 1 97 n/a 99 n/a 10 89 88 88 90 20 79 69 76 69

FIG. 9 is a plot of the area-normalized second derivative FTIR spectraof pressure-treated (FIG. 9A) thermally-induced and (FIG. 9B) guanidinehydrochloride-induced aggregates of rhIFN-γ for pressure-treatmentsconducted at 10 and 20 mg/mL. Due to the preparation and samplecollection times associated with the FTIR technique, FTIR spectra werecollected within three hours of depressurization. For comparativepurposes, the second derivative FTIR spectrum of native rhIFN-γ issuperimposed on both windows of FIG. 9. As was the case with the UVspectroscopic results, the recovery of native secondary structure,measured qualitatively by FTIR, is aggregate-form independent, as thesecond derivative FTIR spectra at 10 and 20 mg/mL are the same for bothaggregate forms, respectively. However, the recovery of native-likesecondary structure is dependent on the protein concentration duringpressure treatment. There is substantial reduction in the intermolecularβ-sheet bands near 1620 and 1695 cm⁻¹ with pressure treatment for boththe 10 and 20 mg/mL samples. But, the reduction of these intermolecularβ-sheet bands to levels observed in the native spectrum is nearlycomplete for the 10 mg/mL spectra, while significant bands are stillpresent near 1620 and 1695 cm⁻¹ in each of the 20 mg/mL spectra. Asmeasured by second derivative FTIR, the samples pressure-treated at 10mg/mL recover all of the α-helix seen in the native structure (band near1656 cm⁻¹). The recoveries of native secondary structure, as measured byarea of overlap (Kendrick et al., 1996), are ca. 90 and 70% for the 10and 20 mg/mL pressure-treatments, respectively. Table 2 compares therecovery of native structure by FTIR, as measured by area of overlap,with the recovery of native structure by second derivative UVspectroscopy. The percent recovery of native-like characteristics, asmeasured by UV and FTIR, are in reasonable agreement for both the 10 and20 mg/mL pressure-treated samples, as the reproducibility of each methodis ca. 5%.

Stability of pressure-treated aggregates: Physical characterization.After initial analysis following pressure treatment, thepressure-treated aggregate samples, aggregate controls and nativecontrols were placed at 4° C. Two weeks after pressure treatment, the 10and 20 mg/mL pressure-treated samples of both the thermally- andguanidine hydrochloride-induced aggregates had formed colorless,transparent gels, similar to the thermally-induced aggregates that werenot pressure-treated. However, the viscosities of these samples (judgedqualitatively) were noticeably lower than the viscosities observed forthe thermally-induced aggregate controls. Qualitative time-dependentdifferences between the aggregates that were pressure-treated at 1 mg/mLwere not apparent.

GEMMA and SEC were performed on the pressure-treated aggregates that hadbeen diluted to 1 mg/mL in succinate buffer immediately after pressuretreatment and subsequently stored for two weeks at 4° C. Results fromGEMMA and SEC for the pressure-treated samples were compared to theliquid and aggregate controls. There were no observed effects, obtainedeither by GEMMA or SEC, with starting aggregate type or refoldingprotein concentration. As measured by GEMMA and SEC, thepressure-treated aggregates contained only two species, which wereidentified as monomer and dimer, and there were no detected higher-orderaggregates in any of the pressure-treated aggregate samples or nativecontrols. GEMMA analysis of the thermally-induced aggregate controlshowed a large (greater than 95% by mass) population of aggregates (FIG.3) (again, GEMMA analysis was not performed on the guanidinehydrochloride-induced control).

A summary of the results between the native control and pressure-treatedaggregates for GEMMA and SEC are presented in Tables 1A and 2B,respectively. Since there were no observed effects with startingaggregate type or refolding protein concentration, the data for thepressure-treated aggregates in Table 1 have been averaged over startingaggregate type and refolding protein concentration. The two peaks in thepressure-treated size distributions, as measured by GEMMA, were centeredat 4.6 and 5.7 nm, which were larger in EM diameters than theirrespective counterparts in the distribution of the control (monomer anddimer). Additionally, by GEMMA, a greater mass percent monomer wasobserved in the refolded aggregates compared to the native control. BySEC, the elution time for the dimer was shorter for the pressure-treatedaggregates than for the control, but there was no measured difference inelution time of the monomer. SEC observed no measurable differences inthe mass percent monomer between the control and the pressure-treatedaggregates. Since GEMMA and SEC data conflicted regarding an increase inmass percent monomer, the data do not support a conclusion of anincreased mass percent monomer in the pressure-treated aggregate samplesrelative to the control.

For both the GEMMA and SEC data, MW estimations for monomer and dimer inthe pressure-treated aggregate and native control samples were made fromthe calibration curves made with proteins of known MW and these MWestimations are presented in Table 1. The MW estimations for the monomerand dimer of the control samples were accurate by both GEMMA and SEC, asthe known MW of rhIFN-γ monomer and dimer are 16.45 and 32.9 kD,respectively. The MW estimates for the dimer of the pressure-treatedaggregate samples were larger by both GEMMA and SEC than the dimer MWestimate for the control samples. The increase in apparent MW of thedimer is believed to result from an improperly folded, expanded statedimer. The apparent MW for the monomer was larger by GEMMA, butunchanged by SEC, relative to the control.

Stability of pressure-treated aggregates: Structural characterization.Two weeks after the pressure refolding was performed, the samples thathad been immediately diluted to 1 mg/mL upon depressurization wereremoved from storage at 4° C. and analyzed by UV. Concentrationlimitations prohibited analysis of these samples by FTIR. FIG. 10 is aplot of the second derivative UV spectra of thermally-induced,pressure-treated aggregates that were pressure-treated at 1, 10 and 20mg/mL. For reference, the second derivative UV spectrum of nativerhIFN-γ is superimposed on FIG. 10. The spectrum of the 1 mg/mLpressure-treated aggregate sample was the same as the spectrum takenimmediately following the pressure treatment and showed essentially fullrecovery of native-like characteristics. However, the spectra of the 10and 20 mg/mL pressure-treated aggregate samples were changed from therespective spectra taken immediately following pressure treatment (FIG.8). Following storage at 1 mg/mL and 4° C. for two weeks, the recoveriesof native structure, as measured by the height of the extremum near 286nm, were 100, 97 and 94% for the 1, 10 and 20 mg/mL thermally-induced,pressure-treated aggregates, respectively. The recoveries of nativestructure for the 10 and 20 mg/mL thermally-induced, pressure-treatedaggregates increased from 89 and 79% (Table 2) measured immediatelyafter pressure treatment, respectively. As was the case immediatelyafter pressure-treatment, the extent of recovery of the native secondderivative UV spectrum two weeks after pressure treatment wasaggregate-form independent. The second derivative UV spectra ofguanidine hydrochloride-induced, pressure-treated aggregate samples arenot presented, but the recoveries of native structure, as measured bythe height of the extremum near 286 nm, were 100, 97 and 95% for the 1,10 and 20 mg/mL samples, respectively. The recoveries of nativestructure, as measured by second derivative UV, after storage at 1 mg/mLand 4° C. show a decreasing trend with protein concentration duringrefolding, but the differences in recoveries are within the accuracy ofthe method.

Concentration dependence on recovery of native dimer. The recovery ofnative dimer from aggregates showed interesting protein concentrationdependence. UV and FTIR spectroscopic data taken soon after therefolding protocol was completed indicate a strong concentrationdependence on the extent of recovery. Additionally, the secondderivative FTIR spectra taken after pressure treatment showed clearsigns of aggregation, particularly for the samples refolded at 20 mg/mL(the presence of the intermolecular β-sheet bands at 1620 and 1695 cm⁻¹in FIG. 9). Further, after two-weeks of storage at 4° C., the undiluted10 and 20 mg/mL pressure-treated aggregate samples showed macroscopicevidence of aggregation by the visible increases in viscosities of thesesamples. However, the same pressure-treated samples, which had beendiluted to 1 mg/mL immediately after pressure treatment, showed no signsof aggregates by GEMMA or SEC, were indistinct from the liquid controlby TEM and showed nearly full recovery of native structure by UVanalysis, regardless of protein concentration during refolding. Toexplain this interesting protein concentration dependence, it isnecessary to understand the pressure effect on and the aggregationpathway of rhIFN-γ.

Hydrostatic pressure is known to dissociate oligomeric proteins (Gross &Jaenicke 1994; Silva & Weber 1993) and has been shown by the inventorsto dissociate rhIFN-γ in the pressure region employed here fordissolution of aggregates. The in situ UV data collected during therefolding protocol indicated that after ca. one hour at 100 MParefolding was essentially complete. The in situ UV data were collectedat 1 mg/mL, but not at 10 and 20 mg/mL due to the absorbance limitationof the technique. Therefore, the refolding rate as a function ofconcentration at 100 MPa is not known. If the rate of refolding at 100MPa is reduced at higher protein concentrations, then substantialmonomer may have been present in the 10 and 20 mg/mL samples after the1.5 hour refolding time at 100 MPa. The inventors showed secondderivative UV spectra of the 10 and 20 mg/mL samples collectedimmediately after depressurization (FIG. 8) are consistent withincreased monomer concentration. Further, it has been established by theinventors that rhIFN-γ aggregates via the monomer, and, therefore,presence of monomeric rhIFN-γ upon depressurization is expected to leadto aggregation. Thus, it is likely that the concentration dependenceobserved (immediately after the pressure treatment) in the secondderivative UV spectra of pressure-treated aggregates (FIG. 8) reflectsan increased monomer population at increased protein concentrations.

So, for the 10 and 20 mg/mL pressure-treated aggregate samples, why isaggregation observed in the undiluted samples and not in the samplesthat were diluted to 1 mg/mL rhIFN-γ? The answer lies in therelationship between protein aggregation and protein folding. Becauseprotein aggregation proceeds through a folding intermediate, aggregationand folding are competitive processes (Betts et al., 1997; Clark et al.,1999; Fink, 1998). Protein refolding steps are generally first-orderprocesses (Clark et al., 1999), but the inventors have determined thataggregation of rhIFN-γ in the absence of high electrolyte concentrationsis second-order in protein concentration. If the rate-limiting step ofrhIFN-γ folding is of lower order than two (e.g. first-order), thenlower protein concentrations will favor refolding over aggregation. Itis reasonable to envision the refolding kinetics of rhIFN-γ to befirst-order when the intertwined nature of the dimer (Ealick et al.,1991) is considered. If the rate-limiting step in the acquisition ofdimer is not the rate of collision of monomers, but the rate of assumingthe correct conformation that allows the refolding to occur, thenfirst-order kinetics would be observed. As well, lowering thetemperature may contribute to the inhibition of aggregation andacquisition of dimer, as the aggregation reaction has a large activationenergy, but the association reaction leading to dimer is relativelyinsensitive to temperature (Boteva et al., 1996). Thus, lowertemperatures favor the refolding reaction over aggregation.

Example 3 Lysozyme Refolding

Pressure was generated using high-pressure nitrogen (40 MPa) connectedto a 10-fold hydraulic intensifier (High Pressure Equipment Company,Erie, Pa.). One mg/ml suspensions of lysozyme aggregates in 50 mM Tris(pH 8.0, at 24° C.), varying amounts of GdnHCl, 2 mM DTT and GSSG at thedesired final ratio (GSSG:GSH; an optimal ratio was 1:1) were preparedin heat-sealed bulbs of SAMCO® transfer pipets, sealed within a bag ofwater, and placed into a to a 2 liter cloverleaf reactor rated to 200MPa and filled with oil. Samples were slowly pressurized (over 20minutes) to final desired pressure to minimize pressurization-inducedheating. Depressurization was conducted in 20 MPa increments, with eachdepressurization step requiring approximately 5 minutes. Samples wereincubated at each intermediate pressure during depressurization for 15minutes, yielding an overall depressurization rate of 1 MPa/minute (10bar/minute). Thermal transients caused by pressure-induced heating wereminimal (<2° C.) at this pressurization rate, as monitored bythermocouples mounted in the pressure vessel. Unless otherwise stated,all pressure experiments were performed at 24° C.

Example 4 Bikunin Refolding

Refolding studies were conducted on bikunin, a 170 amino acid proteinwith six disulfide bonds. This protein often forms an aggregate duringfermentation. The aggregate is composed of non-native disulfide bondsand is an oligomer of four to eight monomers. Experiments were conductedto determine if high hydrostatic pressure (1000-3000 bar) could be usedto refold these disulfide scrambled protein aggregates.

Aggregated bikunin was placed in sealed syringes and held under pressureat various reactor conditions. Pressure, pH, redox conditions,temperature and depressurization rate were all controlled. As a basecase, the following conditions were used: 1 mg/ml sample of aggregatedbikunin was held at 200 MPa, 25C., in 4 mM oxidized glutathione (GGSG),2 mM dithiolthreitol (DTT) for 16 hours. The samples were thendepressurized by 10 MPa every 30 minutes until a pressure of 100 MPa wasreached. The samples were further depressurized 25 MPa every fifteenminutes until 0.1 MPa was reached. After pressurization, the sampleswere analyzed with size exclusion chromatography (SEC) to determine therefolding yield. A calibration curve was used to ensure that massbalance was maintained during experimentation. Samples were also sent toa secondary lab for analysis through reverse phase chromatography (RP)and activity assay. These secondary studies showed that SECover-estimated the refolding yield by about 10% due to the presence ofnon-active monomeric proteins. SEC was still used as an optimizationtool, however final refolding yields were confirmed with an activityassay.

A refolding yield of 45% +/−4% was obtained as measured by activityassay at the “base case” conditions described above. A rapidoptimization of refolding conditions was obtained by varying solutionconditions around the “base case” conditions. An unsophisticatedalgorithm was used; more sophisticated designed experiments well-knownto industrial scientists would likely yield more optimal conditions withfewer experiments. However, even using the simple technique of varyingredox conditions, temperature, pressure and depressurization rateseparately around the base case conditions, a reasonable optimum wasquickly found.

Oxidized and reduced glutathione can be used to control the redoxconditions within the refolding solution. These compounds are needed tobreak non-native disulfide bonds and reform native disulfides.Typically, optimum redox conditions exist when the total. glutathioneconcentration is between 6-16 mM, with ratios of reduced to oxidizedglutathione specific to the system, often between 1 and 3. Samples weremade with varying redox conditions and tested at three differentpressure conditions to determine the effect on refolding yield. Theratio of the monomeric SEC peak to the aggregated SEC peak was used tomeasure the effectiveness of refolding. The results are shown in FIG.11. For these studies, the most optimum case was found to be 4 mMoxidized glutathione, 2 mM DTT at 2000 bar.

The glutathione shuffling system was necessary to obtain any significantrefolding yield, as would be expected for a protein aggregate containingnon-native disulfide bonds. Pressure and DTT alone were not effective.Further optimization of the redox conditions could potentially be done;however, the yields obtained in 4 mM GGSG, 2 mM DTT at 2000 bar wereadequate enough to determine the effects of the remaining refoldingconditions.

The effect of refolding temperature was evaluated. Samples were held at12 hours at varying refolding temperatures, then brought to roomtemperature and held for an additional twelve hours. Pressures of 2000bar and the previously optimized redox conditions were used. Refoldingwas found to be maximal at 25° C. These results are shown in FIG. 12.

The effect of refolding pressure was evaluated. Samples were held at 16hours at varying refolding pressures, and then slowly depressurized overan eight-hour period. The samples were held at room temperature, withthe redox conditions previously used. 2000 bar was found to be theoptimum refolding pressure, with refolding yield decreasing at both 1000bar and 3000 bar. These results are shown in FIG. 13.

Protein monomer was subjected to rapid and slow depressurization ratesto determine the impact of depressurization of protein yield. Sampleswere held at 2000 bar, 25° C., for 16 hours in the standard redoxconditions (4 mM GGSG, 2 mM DTT). One set of samples was depressurizedover a thirty second period. The second set of samples was depressurizedaccording to the procedure described in the experimental methods(samples were depressurized by 10 MPa every 30 minutes until a pressureof 100 MPa was reached, then further depressurized 25 MPa every fifteenminutes until 0.1 MPa was reached). The samples that were depressurizedslowly lost 21% +/−3% of the monomer to aggregates. This value was muchlower than the 59% loss +/−16% when the sample was depressurizedquickly. This study verified the need to use slow depressurization overa period of hours to ensure that the proteins maintain their nativeconformation.

Example 5 aVEGF Refolding

High pressure refolding studies were conducted on non-native dimericaggregates of an antibody against human vascular endothelial growthfactor (aVEGF), a full-length, disulfide binded, glycosylated,heterotetrameric antibody that undergoes irreversible dimerizationduring production in chinese hamster ovary (CHO) cell culture. Refoldingstudies were conducted to determine if high hydrostatic pressure couldbe used to obtain native aVEGF monomers. Additionally, experiments wererun to determine the effect of temperature on refolding yield.

Experiments were conducted to determine the refolding yield of samplesheld at 2000 bar at three different temperatures. The experimentalprocedure is as follows: aVEGF (22% aggregate, 78% monomer) was dilutedto a concentration of 1 mg/ml in 25 mM 2-(4-Morpholino)-ethane sulfonicacid (MES) buffer at pH 6.0. Each sample was made of 0.4 ml of the MESprotein solution (1 mg/ml) loaded into a sealed syringe. The syringe isrequired to ensure proper pressure transfer to the sample. Three sampleswere created for each condition and placed into pressure vessels at roomtemperature. The samples were pressurized to 2000 bar and the pressurevessel temperature adjusted to 0° C., 25° C., or 50° C. The pressure wasmonitored during this time to maintain 2000 bar despite temperaturechanges. The samples were held at the desired temperature and pressurefor sixteen hours, cooled or warmed to room temperature, anddepressurized. Depressurization was done by 100 bar increments everythirty minutes, with drops of 250 bar every fifteen minutes once 1000bar was reached. The samples were removed from the pressure vessels andstored at 4° C. for two days. Size exclusion chromatography (SEC) wasused to determine the mass fractions of monomers and dimers.

Three samples were tested at 0° C., 25° C., and 50° C. and 2000 barthrough the procedure described above. Refolding yield (RY %) wascalculated from the SEC results using the following equation:

${{RY}\mspace{14mu}\%} = {\frac{M_{f} - M_{i}}{1 - M_{i}}*100}$where Mf=final monomer fraction and Mi=initial monomer fraction.

Temperature was found to influence the refolding yield. These studiesshowed that a refolding yield of 29% (+/−1%) at 50° C. could beachieved. This is shown in FIG. 14.

Example 6 High Pressure Refolding of GCSF Inclusion Bodies

Concentrated, purified inclusion bodies supplied from Amgen, Inc.(Thousand Oaks, Calif.) were suspended in refolding buffer (50 mMTris-HCl, 1 mM EDTA, 0.1% NaN₃) containing varying concentrations ofguanidine hydrochloride (GdnHCl). Triplicate samples were pressurized to2000 bar and incubated for 24 hours. Pressure was released by decreasingthe vessel pressure in 200 bar increments. Samples were held at eachincremental pressure for 15 minutes.

Upon complete depressurization, samples were centrifuged at 13,000 g for15 minutes. The resulting supernatant was analyzed with the Pierce® TPAtotal protein assay. Bovine serum albumin (BSA) standards were used tocalibrate the assay. The results are shown in FIG. 15.

All of the COMPOSITIONS, METHODS and APPARATUS disclosed and claimedherein can be made and executed without undue experimentation in lightof the present disclosure. While the compositions and methods of thisinvention have been described in terms of preferred embodiments, it willbe apparent to those of skill in the art that variations may be appliedto the COMPOSITIONS, METHODS and APPARATUS and in the steps or in thesequence of steps of the method described herein without departing fromthe concept, spirit and scope of the invention. More specifically, itwill be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

XII. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

U.S. Pat. No. 4,652,630

U.S. Pat. No. 4,659,568

U.S. Pat. No. 4,677,196

U.S. Pat. No. 4,766,224

U.S. Pat. No. 4,923,967

U.S. Pat. No. 4,929,700

U.S. Pat. No. 4,985,544

U.S. Pat. No. 5,023,323

U.S. Pat. No. 5,064,943

U.S. Pat. No. 5,077,392

U.S. Pat. No. 5,109,117

U.S. Pat. No. 5,162,507

U.S. Pat. No. 5,410,026

U.S. Pat. No. 5,593,865

U.S. Pat. No. 5,605,691

U.S. Pat. No. 5,708,148

U.S. Pat. No. 5,714,371

U.S. Pat. No. 5,728,804

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1. A method of producing biologically active disaggregated protein fromprotein aggregates comprising: (i) providing a protein aggregate; (ii)mixing said protein aggregate with a reducing agent in an amountsufficient to reduce disulfide bonds therein; (iii) subjecting themixture of step (ii) to increased pressure of about 0.25 kbar to about12 kbar, whereby said protein aggregate dissolves and/or disaggregates;(iv) dialyzing the mixture under pressure, whereby said reducing agentis removed and disulfide bonds reform; and (v) removing the dissolvedand/or disaggregated protein from increased pressure, whereby saidprotein refolds such that biological activity is retained.
 2. The methodof claim 1, further comprising the step of agitating said proteinaggregate and/or dissolved protein and/or disaggregated protein toenhance dissolution and/or disaggregation and/or refolding.
 3. Themethod of claim 1, further comprising subjecting said protein aggregate,prior to refolding, to a temperature of about 30° C. to about 125° C. 4.The method of claim 3, wherein said method does not utilize a chaotropicagent.
 5. The method of claim 1, wherein said reducing agent isdithiothreitol, glutathione, dithioerythritol, or β-mercaptoethanol. 6.The method of claim 1, wherein said increased pressure comprises about500 atmospheres to about 10,000 atmospheres.
 7. The method of claim 1,wherein steps (ii)-(v) are performed in about 3 hours to about 12 hours.8. The method of claim 7, wherein steps (ii)-(v) are performed in about6 hours.
 9. The method of claim 1, wherein steps (ii) and (iii) areperformed in the presence of a chaotropic agent, and said method furthercomprises the step of removing said chaotropic agent.
 10. The method ofclaim 8, wherein said chaotropic agent is guanidine, guanidine sulfate,guanidine hydrochloride, urea, thiocyanate, sarcosyl, sodium dodecylsulfate, or sodium octyl sulfate.
 11. The method of claim 1, whereinsaid protein aggregate comprises inclusion bodies, soluble and insolubleprecipitates, soluble non-native oligomers, gel, fibrils, films,filaments, protofibrils, amyloid deposits, plaques, or dispersednon-native intracellular oligomers.
 12. A method of producingbiologically active disaggregated protein from protein aggregatescomprising: (i) providing a protein aggregate; (ii) subjecting the saidprotein aggregate to increased pressure of about 0.25 kbar to about 12kbar, and high temperature of about 30° C. to about 125° C., wherebysaid protein aggregate dissolves and/or disaggregates; and (iii)removing the dissolved and/or disaggregated protein from increasedpressure and high temperature, whereby said protein refolds such thatbiological activity is retained.
 13. The method of claim 12, whereinsaid method does not include use of a chaotropic agent.
 14. The methodof claim 12, further comprising the step of agitating said proteinaggregate and/or dissolved protein and/or disaggregated protein toenhance dissolution and/or disaggregation and/or refolding.
 15. Themethod of claim 12, further comprising mixing said protein aggregatewith a reducing agent in an amount sufficient to reduce disulfide bondstherein.
 16. The method of claim 15, further comprising dialyzing saidmixture under pressure, whereby said reducing agent is removed anddisulfide bonds reform.
 17. The method of claim 15, further comprisingremoving said reducing agent by diafiltration or ultrafiltration. 18.The method of claim 15, further comprising negating the effect of saidreducing agent by addition of an oxidizing agent.
 19. The method ofclaim 15, wherein said reducing agent is dithiothreitol, glutathione,dithioerythritol, or β-mercaptoethanol.
 20. The method of claim 12,wherein said increased pressure comprises about 0.5 kbar to about 10kbar.
 21. The method of claim 12, wherein steps (ii)-(iii) are performedin about 3 hours to about 12 hours.
 22. The method of claim 21, whereinsteps (ii)-(iii) are performed in about 6 hours.
 23. The method of claim12, wherein said protein aggregate comprises inclusion bodies, solubleand insoluble precipitates, soluble non-native oligomers, gel, fibrils,films, filaments, protofibrils, amyloid deposits, plaques, or dispersednon-native intracellular oligomers.
 24. The method of claim 12, whereinsteps (ii) and (iii) are performed in the presence of a chaotropicagent, and said method further comprises the step of removing saidchaotropic agent.
 25. The method of claim 24, wherein said chaotropicagent is guanidine, guanidine sulfate, guanidine hydrochloride, urea,thiocyanate, sarcosyl, sodium dodecyl sulfate, or sodium octyl sulfate.26. A method of producing biologically active disaggregated protein fromprotein aggregates comprising: (i) providing a protein aggregate; (ii)mixing said protein aggregate with a reducing agent in an amountsufficient to reduce disulfide bonds therein; (iii) subjecting themixture of step (ii) to increased pressure of about 0.25 kbar to about12 kbar, high temperature of about 30° C. to about 125° C., andagitation, whereby said protein aggregate dissolves and/ordisaggregates; (iv) removing or neutralizing said reducing agent,whereby disulfide bonds reform; and (v) removing the dissolved and/ordisaggregated protein from increased pressure and high temperature,whereby said protein refolds such that biological activity is retained.27. The method of claim 26, wherein said method does not include use ofa chaotropic agent.
 28. The method of claim 26, further comprisingdialyzing said mixture under pressure, whereby said reducing agent isremoved and disulfide bonds reform.
 29. The method of claim 26, furthercomprising removing said reducing agent by diafiltration orultrafiltration.
 30. The method of claim 26, further comprising negatingthe effect of said reducing agent by addition of an oxidizing agent. 31.The method of claim 26, wherein said reducing agent is dithiothreitol,glutathione, dithioerythritol, or β-mercaptoethanol.
 32. The method ofclaim 26, wherein said increased pressure comprises about 0.5 kbar toabout 10 kbar.
 33. The method of claim 26, wherein steps (ii)-(iii) areperformed in about 1 hour to about 12 hours.
 34. The method of claim 33,wherein steps (ii)-(iii) are performed in about 6 hours.
 35. The methodof claim 26, wherein said protein aggregate comprises inclusion bodies,soluble and insoluble precipitates, soluble non-native oligomers, gel,fibrils, films, filaments, protofibrils, amyloid deposits, plaques, ordispersed non-native intracellular oligomers.
 36. The method of claim26, wherein steps (ii) and (iii) are performed in the presence of achaotropic agent, and said method further comprises the step of removingsaid chaotropic agent.
 37. The method of claim 36, wherein saidchaotropic agent is guanidine, guanidine sulfate, guanidinehydrochloride, urea, thiocyanate, sarcosyl, sodium dodecyl sulfate, orsodium octyl sulfate.
 38. A method of refolding denatured proteincomprising: (i) providing a denatured protein composition; (ii) mixingsaid protein composition with a reducing agent in an amount sufficientto reduce disulfide bonds therein; (iii) subjecting the mixture of step(ii) to increased pressure of about 0.25 kbar to about 12 kbar; (iv)dialyzing the mixture under pressure, whereby said reducing agent isremoved and disulfide bonds reform; and (v) removing the proteincomposition from increased pressure, whereby said protein refolds suchthat biological activity is retained.
 39. A method of refoldingdenatured protein comprising: (i) providing a protein composition; (ii)subjecting the said protein composition to increased pressure of about0.25 kbar to about 12 kbar, and high temperature of about 30° C. toabout 125° C.; and (iii) removing the protein composition from increasedpressure and high temperature, whereby said protein refolds such thatbiological activity is retained.
 40. A method of refolding denaturedprotein comprising: (i) providing a protein composition; (ii) mixingsaid protein composition with a reducing agent in amount sufficient toreduce disulfide bonds therein; (iii) subjecting the mixture of step(ii) to increased pressure of about 0.25 kbar to about 12 kbar, hightemperature of about 30° C. to about 125° C., and agitation; (iv)removing or neutralizing said reducing agent, whereby disulfide bondsreform; and (v) removing the protein composition from increased pressureand high temperature, whereby said protein refolds such that biologicalactivity is retained.
 41. A method of rendering a protein aggregatesusceptible to dissolution and/or disaggregation by detergents and/orincreased temperature comprising subjecting said protein aggregate tohigh pressure of about 0.25 kbar to about 12 kbar in combination withone or more of detergents and/or increased temperature.
 42. A method ofproducing biologically active disaggregated protein from proteinaggregates comprising: (i) providing a protein aggregate; (ii)subjecting the mixture of step (i) to increased pressure of about 0.25kbar to about 12 kbar, high temperature of about 30° C. to about 125° C.and agitation, whereby said protein aggregate dissolves and/ordisaggregates; (iii) altering the pH of the mixture of step (ii) bydialysis; and (iv) removing the dissolved and/or disaggregated proteinfrom increased pressure and high temperature, whereby said proteinrefolds such that biological activity is retained.